Visualization of Knee Joint Degeneration after Non-invasive ACL Injury in Rats

The ACL is a band-like structure of dense connective tissue that arises from the anterior intercondylar space of the tibia and extends superiorly and laterally to the posterior aspect of the lateral condyle of the femur. Structurally, the ACL serves as both a passive stabilizer of the knee, working in concert with other ligaments as well as thigh musculature to help control the joint during dynamic movement. The ACL is the primary restraint to anterior tibial displacement and plays an essential role in maintaining knee joint stability. Beyond structural support, the ACL also acts as a pathway for neural information between the knee joint and central nervous system. The greatest stress on the ACL occurs when the knee is near extension, and it is during this time that the ACL is at the highest risk of injury.

The ACL is the most commonly injured knee ligament during sport and work-related activities. Non-contact ACL injuries account for nearly 70% of all ACL injuries, and they occur when a person generates sufficient forces and/or moments at the knee that leads to excessive loading of the ACL. Although the mechanism of non-contact ACL injury has been investigated using a variety of research models (prospective, retrospective, observational, in vivo and in vitro), a direct determination of how injury occurs remains elusive. ACL reconstruction is often performed by surgically inserting a portion of the individuals hamstring or patellar tendon into the area of the ACL. The aim of surgical reconstruction is to maximize knee stability and functional capacity that were lost following the injury. Surgical reconstruction facilitates a safe return to sport and promotes long-term knee joint health. However, despite the best efforts of clinicians and researchers, nearly two-thirds of patients with a reconstructed ACLs reconstructed patients do not return back to activity at 12 months post-reconstruction and more than 50% of ACL reconstructed knees have radiographic signs of PTOA 5-14 years after injury.

Animal models provide both a practical and clinically relevant way to study the natural history and response of treatment to joint health. Importantly the knee of a rat has similar anatomy and function to knees in humans, which makes the rat knee a useful model to study PTOA after ACL injury. To improve the clinical translation of our results, we have recently developed a novel non-invasive model of ACL injury, where the ACL is ruptured through a single load of tibial compression. This injury closely replicates injury conditions relevant to humans and is highly reproducible.

The load device consists of two custom-built loading platforms (Figure 1); the top knee stage is rigidly mounted to a linear actuator (DC linear actuator L16-63-12-P, Phidgets, Alberta, CA) that positions the right hindlimb in 30°^1-3 of dorsiflexion and 100°¹ of knee flexion while providing room for anterior subluxation of the tibia relative to the femur; the bottom stage holds the flexed knee and is mounted directly above a load cell (HDM Inc., PW6D, Southfield, MI). During the injury, rats are anesthetized and then the right hindlimb is subjected to single load of tibial compression at a speed of 8 mm/s.¹ ACL injury is noted by a release of compressive force during injury that is monitored via a custom program (LabVIEW, National Instruments, Austin, TX). Post-injury, ACL rupture is clinically confirmed by the Lachman's test, where the femur is secured while an anterior force is applied to the tibia. Excessive anterior tibial translation indicates ACL deficiency. The ACL injured hindlimb can then be extended and secured in a custom 3D printed device to visualize knee joint degeneration. Images are acquired to characterize changes in trabecular structure related to PTOA development.⁴

Figure 1: Tibial compressive load causing isolated non-invasive ACL injury.

Smaller trabecular number, reduced trabecular thickness and greater trabecular spacing, all hallmark characteristics of PTOA onset, were evident 4 weeks after the non-invasive ACL tear (Table 1 and Figure 3). An image of a dissected ACL of healthy limb versus an acute injured limb is shown in Figure 5. The novel non-invasive model of ACL injury, where the ACL is ruptured through a single load of tibial compression, was able to produce an isolated proximal tear of the ACL.

Figure 3: 3-D reconstructed μCT image of an acute ACL-injury (left) and 4 weeks post-ACL injury (right) in a rat. 

Table 1: Characteristic measurements of PTOA onset.

Figure 4: Image of an acute injured ACL-limb (left) and image of intact, healthy ACL (right).

This video demonstrates how a linear actuator can be used to produce an isolated non-invasive ACL rupture in rats. This injury closely replicates injury conditions relevant to humans and is highly reproducible. To overcome several of the major limitations of traditional OA staining techniques, this method uses µCT to quantify whole joint degeneration and trabecular structure.

Evidence-based interventions to improve musculoskeletal rehabilitative outcomes is a highly significant area that has changed little in the past two decades, even though significant advances in basic biology have suggested that alterations to rehabilitation protocols are long overdue. The issue is that classically rehabilitation specialists have used anecdotal reports to shape clinical practice rather than basic science to provide informed hypotheses that are tested in model organisms before translation to the clinic. The procedures described here provide scientists with a method to closely replicate a traumatic joint injury that is relevant to humans and use µCT to track the progression of joint health.

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