Poster 254: Mechanical Response of ACL to Submaximal Fatigue Loading
Authors: Loflin Benjamin , Colglazier Kaitlyn , Schlecht Stephen 
Published: July 2022

Objectives: It has been previously reported that the ACL increases in size and strength throughout rodent adolescence following both voluntary1 and forced2 endurance running. Here, we expand upon these findings to investigate how the ACL responds to submaximal fatigue loading. The ACL of sexually mature mice was subjected to 3 days of in vivo repetitive loading with an intervening day for recovery. We hypothesized that direct repetitive loading would should a greater positive change in ACL mechanical properties than what was previously observed following endurance running. To test this hypothesis, we designed a custom loading fixture to apply repetitive ACL loading cycles up to a predetermined percentage of the ACL failure load while approximating the knee kinematics of a jump-landing with a pivot shift. Methods: With Institutional approval, 20 C57BL/6J 10-week-old female mice underwent ACL fatigue loading across 3 days with an intervening recovery day following each training session. For each loading session, the right rear leg of the mouse was placed in a custom loading fixture with the knee at 90° flexion and the sole of the foot at 20° inversion to induce a valgus moment across the knee (Fig. 1). Once positioned, the foreleg was internally rotated. Internal tibial rotation combined with a valgus moment across the knee generates maximum peak ACL strain and replicates a well characterized clinical ACL injury mechanism3. Based on our previous in vivo work we chose two ACL ultimate tensile strength (UTS) loading percentages that represent low and moderate loads to repetitively fatigue the ligament. An in vivo 60% ACL UTS reliably generates collagen triple helix degradation followed by a proteoglycan response (Fig. 2), while no such changes occur at an in vivo 30% ACL UTS. Therefore, for each training session the ACL was cyclically loaded in vivo between 30% and 60% of ACL UTS for 440 cycles at a loading rate of 0.75 mm/s. After the third session mice were allowed to recover for 72 hrs. Following this, the tested and age-matched control ACLs were ruptured in vivo using the same knee kinematics at a loading rate of 2.7 N/s. All ACLs failed interstitially with there being no visible damage to other supporting knee ligaments or menisci. The resulting load-displacement curves were analyzed using custom MatLab code to quantify the mechanical properties of the fatigued and non-fatigued ACLs. In vivo measures quantified included ACL UTS, stiffness (S), total yield strength (TYS) and post-yield displacement (PYD). Data were analyzed using a two-way ANOVA with final body weight included as a covariate in order to determine the mechanical effect that the 3 days of submaximal fatigue loading had on the ACL compared to controls. Results: The submaximal fatigue loading had a significant effect on ACL mechanical properties when compared to controls. Five mice suffered an ACL failure during the fatigue loading experiment, suggesting that 60% ACL UTS may be near the fatigue failure threshold in these mice. The remaining 15 mice that completed the fatigue study showed a 24.0% increase in UTS, a 26.8% increase in S, a 18.5% increase in TYS, and a 130.5% increase in post-yield displacement, compared to controls (Table 1). Conclusions: The outcomes of this study partially confirmed our hypothesis. Three days of direct repetitive loading elicited a greater ACL mechanical response than similar aged female mice following 4 weeks of endurance running. The marked improvement in ACL mechanics following repetitive training was likely due to the greater ACL loads experienced and the time allowed for tissue recovery. Rodent ligaments can turn collagen over in ˜24 hours4. This suggests that the accumulation of degenerated ACL collagen triple helix structures was repaired during recovery for 75% of the mice. However, it also suggests that for some mice, ACL collagen degeneration was not repaired but rather accumulated and propagated hierarchically to the fibril and fiber level, resulting in tissue failure, as was previously observed in human cadaver knees subjected to submaximal ACL fatigue loading5. This study further elucidates how the ACL responds to loading during adolescence. We have demonstrated that systematic repetitive ACL loading in adolescent mice can generate a positive mechanical response in the ACL. However, our findings also suggest that for a subset of mice participating in this training the submaximal fatigue load was too high and/or the duration of the recovery period was insufficient, resulting in catastrophic failure of the ligament prior to study completion. In the future, these failures should be able to be prevented by reducing the maximum load experienced by the ACL, increasing the duration of the recovery period, or both. If translatable to humans, these and future findings may assist clinicans in identifying potential risks for ACL injury related to an individual’s training intensity and help guide clinical inteventions. 1Schlecht et al., J Orthop Res, 2019; 2Cabaud et al., Am J Sports Med, 1980; 3Wojtys et al., J Orthop Res, 2016; 4Sodek, Arch Oral Biol, 1977; 5Chen et al., Am J Sports Med, 2019. [Figure: see text][Figure: see text][Table: see text]