An Adolescent Murine In Vivo Anterior Cruciate Ligament Overuse Injury Model

Abstract

Background: Overuse ligament and tendon injuries are prevalent among recreational and competitive adolescent athletes. In vitro studies of the ligament and tendon suggest that mechanical overuse musculoskeletal injuries begin with collagen triple-helix unraveling, leading to collagen laxity and matrix damage. However, there are little in vivo data concerning this mechanism or the physiomechanical response to collagen disruption, particularly regarding the anterior cruciate ligament (ACL). Purpose: To develop and validate a novel in vivo animal model for investigating the physiomechanical response to ACL collagen matrix damage accumulation and propagation in the ACL midsubstance, fibrocartilaginous entheses, and subchondral bone. Study Design: Controlled laboratory study. Methods: C57BL/6J adolescent inbred mice underwent 3 moderate to strenuous ACL fatigue loading sessions with a 72-hour recovery between sessions. Before each session, randomly selected subsets of mice (n = 12) were euthanized for quantifying collagen matrix damage (percent collagen unraveling) and ACL mechanics (strength and stiffness). This enabled the quasi-longitudinal assessment of collagen matrix damage accrual and whole tissue mechanical property changes across fatigue sessions. Additionally, all cyclic loading data were quantified to evaluate changes in knee mechanics (stiffness and hysteresis) across fatigue sessions. Results: Moderate to strenuous fatigue loading across 3 sessions led to a 24% weaker ( P = .07) and 35% less stiff ( P < .01) ACL compared with nonloaded controls. The unraveled collagen densities within the fatigued ACL and entheseal matrices after the second and third sessions were 38% ( P < .01) and 15% ( P = .02) higher compared with the nonloaded controls. Conclusion: This study confirmed the hypothesis that in vivo ACL collagen matrix damage increases with tissue fatigue sessions, adversely impacting ACL mechanical properties. Moreover, the in vivo ACL findings were consistent with in vitro overloading research in humans. Clinical Relevance: The outcomes from this study support the use of this model for investigating ACL overuse injuries.

Acute Bone Loss and Infrapatellar Fat Pad Fibrosis in the Knee After an In Vivo ACL Injury in Adolescent Mice

“…After 1 week of acclimation, mice were weighed, provided an analgesia, anesthetized, and placed into our validated custom loading fixture (Figure 2) to generate an in vivo ACL injury. 30 Right ACLs were injured and left knees served as the controls. After inducing right ACL injuries, 60 of the 75 mice were block randomized into postinjury cohorts (1, 3, 7, 14, and 28 days after injury) (n = 12/time point).…”

“…(B) Mouse knee position with internal tibial rotation ( θ IR ) and a valgus moment across the knee ( θ M ), while a single axial compressive force ( F ) is applied. Reprinted with permission from Loflin et al 30…”

“…Rigor and reproducibility of hindlimb positioning were validated before the experiment using bilateral radiographs to track hip and knee flexion angles under static loading. 30 Importantly, once compressive loading was initiated, there was no time at which the femoral cup broke contact with the distal femur (Figure 2A), reducing the likelihood of contusing the bone. Maintaining contact also limited joint movement relative to its initial positioning, with no measurable ATT observed for up to ≥60% of the maximum load to failure ( P = .94).…”

“…For these 2 experiments, serial sections from the paired knees of 3 randomly selected mice were chosen from each postinjury cohort (1, 3, 7, 14, and 28 days) (n = 4 sections/knee). Sections were 30 deparaffinized in xylene and then rehydrated in graded alcohols and distilled water. For TRAP (Sigma-Aldrich) and TUNEL (Thermo Fisher) assays, the manufacturerrecommended protocols were followed.…”

Cyclic loading induces anabolic gene expression in ACLs in a load‐dependent and sex‐specific manner

“…3 However, recent studies have provided evidence to suggest that ACLs are susceptible to fatigue failure. Specifically, repetitive subfailure loading has been shown to cause the accumulation of microstructural tissue damage that weakens the ACL 4 and ultimately predisposes the ACL to injury. 5,6 In addition to direct mechanical damage, data from tendons suggest that fatigue loading of ACLs may also induce tissue degeneration that further weakens the tissue and accelerates fatigue failure.…”