Abstract
Residual strain development in biological tissue is believed to result from remodeling in response to repetitive loading. This study hypothesized that differences in in-vivo loading between levels of the bovine tail result in differences in intervertebral disc (IVD) annulus fibrosus (AF) microstructural remodeling. The hypothesis was tested by quantifying tail musculature using clinical computed tomography and tissue microstructure using collagen fiber crimp period, which has previously been correlated with residual strain.
Three bovine tail segments (levels c1 through c6) were imaged using a clinical computed tomography (CT) scanner followed by removal of muscle and harvest of IVDs. The discs were frozen, and transverse cryosections were obtained. Additionally, tangential plane cryosections were obtained from the inner and outer zones of the AF.
Transverse CT slices corresponding to each joint level thresholded for both disc and muscle tissue and analyzed in MATLAB. First, the centroid of the disc image was calculated to use as an origin. Then the disc area and moments of inertia about the flexion extension axis and lateral bending axis were calculated. Total muscle area was then calculated, along with muscle moments of inertia relative to the disc centroid. All muscle parameters were normalized by those of the corresponding disc.
Cryosections were imaged using an inverted light microscope equipped with crossed polarizing filters and a digital camera. A MATLAB routine was used to perform Fourier transform analysis on user selected lines of interest in the transverse micrographs, yielding average fiber crimp period in the inner and outer AF. Micrographs from tangential sections were opened in ImageJ, and fiber orientation angles were measured manually.
Muscle moments of inertia were analyzed using a two-way ANOVA with disc level and axis as dependent variables. Normalized muscle area was analyzed with a one-way ANOVA with disc level as a dependent variable. A two-way ANOVA, with disc level and zone (inner versus outer) was used to analyze collagen fiber crimp period and collagen fiber angle.
Normalized muscle moment of inertia showed significant effects of both level and axis (p < 0 .001), decreasing at distal levels, and being lower about the flexion-extension axis than the lateral bending axis. Normalized muscle cross section showed a visible, but not significant (p=0.0721) decreasing trend with disc level. Fiber crimp period had significant effects of both level and zone (p < 0 .001), and was significantly longer in the outer zone than inner at all levels. Significant decrease in crimp period at distal levels were seen in the outer AF, but not the inner. While fiber angle was significantly (p < 0 .001) higher in the inner AF (36±6.6°) than outer AF (24±3.5°)), there was no significant effect of level.
Fiber crimp period in the AF has previously been correlated with residual circumferential strain, with larger crimp period corresponding to increased residual tension. The present study suggests that at proximal levels of the tail, where peak compressive and bending stresses in the AF (as inferred from normalized muscle area and moments of inertia respectively) are greatest, there is more accumulation of residual strain.
