This study describes the development and application of a novel rat patellar tendon model of mechanical fatigue for investigating the early in vivo response to tendon subfailure injury. Patellar tendons of adult female Sprague-Dawley rats were fatigue loaded between 1–35 N using a custom-designed loading apparatus. Patellar tendons were subjected to Low-, Moderate- or High-level fatigue damage, defined by grip-to-grip strain measurement. Molecular response was compared with that of a laceration-repair injury. Histological analyses showed that progression of tendon fatigue involves formation of localized kinked fiber deformations at Low damage, which increased in density with presence of fiber delaminations at Moderate damage, and fiber angulation and discontinuities at High damage levels. RT-PCR analysis performed at 1- and 3-day post-fatigue showed variable changes in type I, III and V collagen mRNA expression at Low and Moderate damage levels, consistent with clinical findings of tendon pathology and were modest compared with those observed at High damage levels, in which expression of all collagens evaluated were increased markedly. In contrast, only type I collagen expression was elevated at the same time points post-laceration. Findings suggest that cumulative fatigue in tendon invokes a different molecular response than laceration. Further, structural repair may not be initiated until reaching end-stage fatigue life, where the repair response may unable to restore the damaged tendon to its pre-fatigue architecture.

The mechanical and microstructural bases of tendon fatigue, by which damage accumulates and contributes to degradation, are poorly understood. To investigate the tendon fatigue process, rat flexor digitorum longus tendons were cyclically loaded (1–16 N) until reaching one of three levels of fatigue damage, defined as peak clamp-to-clamp strain magnitudes representing key intervals in the fatigue life: i) Low (6.0%–7.0%); ii) Moderate (8.5%–9.5%); and iii) High (11.0%–12.0%). Stiffness, hysteresis, and clamp-to-clamp strain were assessed diagnostically (by cyclic loading at 1–8 N) before and after fatigue loading and following an unloaded recovery period to identify mechanical parameters as measures of damage. Results showed that tendon clamp-to-clamp strain increased from pre- to post-fatigue loading significantly and progressively with the fatigue damage level (p≤0.010). In contrast, changes in both stiffness and hysteresis were significant only at the High fatigue level (p≤0.043). Correlative microstructural analyses showed that Low level of fatigue was characterized by isolated, transverse patterns of kinked fiber deformations. At higher fatigue levels, tendons exhibited fiber dissociation and localized ruptures of the fibers. Histomorphometric analysis showed that damage area fraction increased significantly with fatigue level (p≤0.048). The current findings characterized the sequential, microstructural events that underlie the tendon fatigue process and indicate that tendon deformation can be used to accurately assess the progression of damage accumulation in tendons.

Mechanical overloading is a major causative factor of tendinopathy; however, its underlying mechanisms are unclear. We hypothesized mechanical overloading would damage tendons and alter genes associated with tendinopathy in a load-dependent manner. To test this hypothesis, we fatigue loaded rat patellar tendons in vivo and measured expression of the matrix-degrading enzyme MMP-13 and the inflammatory cytokine IL-1b. We also examined these responses in cultured tenocytes exposed to intermittent hydrostatic pressure in vitro. Additionally, we hypothesized load-induced changes in tenocyte MMP-13 expression would be dependent on expression of IL-1b. In vivo fatigue loading at 1.7% strain caused overt microstructural damage and upregulated expression of MMP-13 and IL-1b, while 0.6% strain produced only minor changes in matrix microstructure and downregulated expression of both MMP-13 and IL-1b. Loading of cultured tenocytes at 2.5 and 7.5 MPa produced comparable changes in expression to those of in vivo tendon loading. Blocking IL-1b expression with siRNA suppressed load-induced both MMP-13 mRNA expression and activity. The data suggest fatigue loading alters expression of MMP-13 and IL-1b in tendons in vivo and tenocytes in vitro in a load-dependent manner. The data also suggest MMP-13 is regulated by both IL-1b-dependent and IL-1b-independent pathways.

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