Anterior cruciate ligament microfatigue damage detected by collagen autofluorescence in situ

Abstract

Purpose Certain types of repetitive sub-maximal knee loading cause microfatigue damage in the human anterior cruciate ligament (ACL) that can accumulate to produce macroscopic tissue failure. However, monitoring the progression of that ACL microfatigue damage as a function of loading cycles has not been reported. To explore the fatigue process, a confocal laser endomicroscope (CLEM) was employed to capture sub-micron resolution fluorescence images of the tissue in situ. The goal of this study was to quantify the in situ changes in ACL autofluorescence (AF) signal intensity and collagen microstructure as a function of the number of loading cycles. Methods Three paired and four single cadaveric knees were subjected to a repeated 4 times bodyweight landing maneuver known to strain the ACL. The paired knees were used to compare the development of ACL microfatigue damage on the loaded knee after 100 consecutive loading cycles, relative to the contralateral unloaded control knee, through second harmonic generation (SHG) and AF imaging using confocal microscopy (CM). The four single knees were used for monitoring progressive ACL microfatigue damage development by AF imaging using CLEM. Results The loaded knees from each pair exhibited a statistically significant increase in AF signal intensity and decrease in SHG signal intensity as compared to the contralateral control knees. Additionally, the anisotropy of the collagen fibers in the loaded knees increased as indicated by the reduced coherency coefficient. Two out of the four single knee ACLs failed during fatigue loading, and they exhibited an order of magnitude higher increase in autofluorescence intensity per loading cycle as compared to the intact knees. Of the three regions of the ACL - proximal, midsubstance and distal - the proximal region of ACL fibers exhibited the highest AF intensity change and anisotropy of fibers. Conclusions CLEM can capture changes in ACL AF and collagen microstructures in situ during and after microfatigue damage development. Results suggest a large increase in AF may occur in the final few cycles immediately prior to or at failure, representing a greater plastic deformation of the tissue. This reinforces the argument that existing microfatigue damage can accumulate to induce bulk mechanical failure in ACL injuries. The variation in fiber organization changes in the ACL regions with application of load is consistent with the known differences in loading distribution at the ACL femoral enthesis.

An Adolescent Murine In Vivo Anterior Cruciate Ligament Overuse Injury Model

“…Thus, to mitigate ACL overuse injuries, we need a greater understanding of how the collagen physiologically, structurally, and functionally responds to repetitive submaximal loading during adolescence. Here, we showed that in vivo submaximal fatigue loading of the ACL in mice reproduces a similar damage signature to that reported in human cadaveric and patient ACLs, 4,8 and by many other research groups across musculoskeletal tissues. 26,28,29,33 Notably, in vivo submaximal fatigue loading generates molecular collagen damage within the ACL, which results in diminished mechanical properties with continued repetitive loading.…”

Overuse Noncontact ACL Injury in Young Athletes: Since We Can’t Completely Fix It, Why Not Prevent It?

“…The exact combination of recovery time, reduced or modified loading, and nutrition that is needed to decrease the potential for sustaining an overuse noncontact ACL injury is not currently known. 8,16 Taken in the aggregate, however, any combination of increased or enhanced recovery, reduced or modified loading, and improved nutrition has high potential to prevent fatigue failure from overuse-related noncontact ACL injury mechanisms. 16 Let's start these discussions.…”

“…Within this scenario, focused attention solely on high volume skill or performance training and competition may get the better of the capacity of the ACL ECM to heal, replacing natural recovery homeostasis with progressive collagen fiber and crosslink degradation, leading to the ligament histopathology that precedes sudden noncontact fatigue rupture. 8,24 For any athlete who has ever experienced this, it remains a memorable event for the rest of their life and often is later recalled as the initial marker that led to the need for more knee surgery.…”

“…Associated with a more wellness-based approach is the fact that, from a medical economics standpoint, society cannot continue to subsidize the growing burden of primary and secondary ACL injury, contralateral knee injuries, and associated surgical and rehabilitation treatment. We have much more to learn, but fatigue failure from overuse is likely associated with many noncontact ACL injuries, and this overuse is likely related to the accumulated training volume associated with early professionalism and specialization in youth and adolescent sports that has replaced what used to be called “play.” 8,13,16…”

“…We have much more to learn, but fatigue failure from overuse is likely associated with many noncontact ACL injuries, and this overuse is likely related to the accumulated training volume associated with early professionalism and specialization in youth and adolescent sports that has replaced what used to be called "play." 8,13,16 Why not prevent what we can't completely fix? Perhaps in 2023, healthcare providers, coaches, event organizers, parents, and youth and adolescent athletes can resolve to better protect the ACL.…”