Accumulation of collagen molecular unfolding is the mechanism of cyclic fatigue damage and failure in collagenous tissues

Abstract

Overuse injuries to dense collagenous tissues are common, but their etiology is poorly understood. The predominant hypothesis that micro-damage accumulation exceeds the rate of biological repair is missing a mechanistic explanation. Here, we used collagen hybridizing peptides to measure collagen molecular damage during tendon cyclic fatigue loading and computational simulations to identify potential explanations for our findings. Our results revealed that triple-helical collagen denaturation accumulates with increasing cycles of fatigue loading, and damage is correlated with creep strain independent of the cyclic strain rate. Finite-element simulations demonstrated that biphasic fluid flow is a possible fascicle-level mechanism to explain the rate dependence of the number of cycles and time to failure. Molecular dynamics simulations demonstrated that triple-helical unfolding is rate dependent, revealing rate-dependent mechanisms at multiple length scales in the tissue. The accumulation of collagen molecular denaturation during cyclic loading provides a long-sought "micro-damage" mechanism for the development of overuse injuries.

Fig. 1. Study overview.

(A) Rat tail tendon fascicles were loaded in creep-fatigue to 40% of the ultimate tensile strength (UTS) until tissue failure. Incremental levels of fatigue were defined as the peak cyclic (creep) strain at 20, 50, and 80% of cycles to failure. (B) To label and quantify denatured collagen, we stained mechanically loaded fascicles with fluorescent CHP, which hybridizes to unfolded collagen α chains. The amount of denatured collagen was quantified on microplate via the fluorescence of bound F-CHP. Computational simulations were used to investigate the potential mechanisms of fascicle- and molecular-level fatigue behavior. (C) Biphasic finite-element simulations were used to study the potential role of fluid flow on strain rate–dependent fatigue behavior at the fascicle level. (D) MD simulations of collagen model peptides were used to identify molecular mechanisms of fatigue damage accumulation and strain rate dependence. GPO, glycine-proline-hydroxyproline.

Fig. 2. Fascicle-level tendon creep and molecular-level mechanical damage to collagen during cyclic creep loading.

(A) All samples exhibited creep behavior, with the sample strain increasing with additional cycles to the same stress level. A triphasic creep pattern was observed at all strain rates; high rates of creep were observed in the initial and final loading cycles, with steady-state creep rate for the majority of the loading duration. While the mean peak cyclic strain was not significantly different between strain rates (P = 0.756; n = 10 per strain rate), there was less variation in the peak cyclic strain at higher strain rates, indicated by the smaller shaded region of SD. (B) Denatured collagen accumulated in the samples throughout the fatigue loading process (P < 0.001; n ≥ 5 per group), with a noticeable increase as early as the strain corresponding to only 20% of cycles to failure. However, the amount of denatured collagen as a function of the normalized number of loading cycles was similar between strain rates (P = 0.179). (C) Plotting the amount of collagen that was mechanically denatured against the peak cyclic strain of the sample reveals the strong correlation of damage with applied strain, without a significant effect of the strain rate of the cyclic loading. These data reveal an apparent damage threshold between 0 and 20% of cycles to failure and between 0 and ~3.8% strain. Mean ± SD in (A) and mean ± SE in (B) and (C).

Fig. 3. Strain rate dependence of fatigue failure and effective material strength.

Strain rate had a significant effect on both (A) the number of cycles to failure (P < 0.001; n = 10 per strain rate) and (B) time to fatigue failure (P = 0.003; n = 10 per strain rate). While samples required more loading cycles to failure at increased strain rates, they failed in less time. This strain rate dependence of both cycles and time to failure demonstrates that tendon creep-fatigue failure is not a pure creep process, which would be dependent only on the time under strain. (C) The monotonic UTS also exhibited significant strain rate dependence (P < 0.001; n = 18 for 0.4% s⁻¹, n = 12 for 1% s⁻¹, and n = 15 for 4 and 40% s⁻¹). The strain rate–dependent UTS may contribute to samples enduring more loading cycles at higher strain rate under the same stress magnitude, as they are being loaded to a lower stress relative to the effective tissue strength. Median ± interquartile range (IQR) with bars representing the range within 1.5 IQR in (A) and (B) and mean ± SD in (C).

Fig. 4. Biphasic rate dependence of tendon mechanics under simulated cyclic loading.

(A) Finite-element simulations predicted tendon creep during cyclic loading with strain at the loading peak reaching an equilibrium around 50 s, which was similar to the transition time from the primary to the secondary phase of fatigue in experiments. The similar equilibrium time between cyclic frequencies revealed that more cycles were required to reach equilibrium at higher frequency. (B) Fluid exudation was predicted to increase with time during cyclic loading, and (C) the lateral contraction (Poisson’s ratio) at the loading peak correspondingly increased. Similar to strain, more cycles were required to reach equilibrium at higher frequencies. Thus, more fluid exuded with each loading cycle at lower frequency, decreasing the material cross-sectional area and increasing the amount of stress on the solid phase. All graphs plot the values at the peak of each loading cycle.

Fig. 5. Simulated fatigue failure of the triple-helix collagen peptide due to shear-dominant loading.

Triple-helix peptides were subjected to repeated cycles of shear-dominant loading between constant force levels, at four different displacement rates. (A to C) Cyclic equilibrium did not exist at any of the displacement rates simulated. Although molecules did not follow the same log-linear correlation of time to failure and strain rate as experiments, the triple helix loaded at 10 m/s or slower exhibited longer times to failure than at 20 m/s. The consistent time to failure at or below a displacement rate of 10 m/s suggests that the failure of individual triple helices is a creep-dominant mechanism, while fascicle-level fatigue failure (i.e., assemblies of molecules) was not entirely explained by creep. avg, average. (D) Triple-helix damage progressed by unraveling and unfolding with catastrophic failure occurring via α-chain pullout. Simulation snapshots correspond to points identified for the simulation in (C).