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.