Mechanical and structural changes to the annulus fibrosus in response to Sub-failure cyclic loading

This study aimed to quantify how repetitive tensile loading alters the mechanical and structural properties of the annulus fibrosus (AF). Mechanical changes were evaluated through a three-step protocol involving pre-damage characterization of dynamic and viscoelastic properties, damage induction using predetermined loading cycles (n=400, 1600, 6400, 12,800) to a specified strain magnitude (11 %, 20 %, 28 %, 44 %), and post-damage characterization of the same properties. Structural changes were assessed by subjecting tissue to damage cycles and staining with hematoxylin and eosin or fluorescing collagen hybridizing peptides (F-CHP). The results showed that damage cycles induced dose-dependent changes in the elastic and viscoelastic responses of the AF, decreasing the tissue's response nearly 100 % of the pre-damage values. Quasi-static distraction to failure revealed that damage cycles influenced the transition strain magnitude, which ranged from 0.11 to 0.31, but did not alter the tissue's ultimate properties. Structural analysis demonstrated cleft formation and collagen fiber uncrimping within the matrix, correlating with the magnitude of loading. However, F-CHP staining revealed no significant differences in denatured collagen fibers between damage groups. Overall, increasing damage parameters significantly decreased the dynamic and viscoelastic properties but did not affect the ultimate properties of the AF. Structural changes indicated disruption of elastic fibers within the AF microstructure without evidence of collagen fiber fractures. These findings provide new insights into the mechanics of healthy and damaged AF tissue, offering a foundational dataset for understanding AF degeneration and injury. STATEMENT OF SIGNIFICANCE: This study investigated the dose-dependent mechanical and structural changes in the annulus fibrosus under sub-failure cyclic loading, addressing a gap in the literature regarding how such loading alters annulus fibrosus properties. By systematically varying strain magnitudes and cycle counts, the work identifies dose-dependent changes in the AF's elastic and viscoelastic behavior, along with structural alterations such as cleft formation and collagen fiber uncrimping. The integration of mechanical testing with histological analysis provides a comprehensive assessment of damage mechanisms in isolated AF tissue. These findings advance the current understanding of AF degradation and fatigue behavior, offering valuable insights for researchers studying spine biomechanics, injury prevention, and interventions aimed at mitigating spinal degeneration.