One of the most common knee injuries is the rupture or tear of the anterior cruciate ligament, also called the ACL, with almost one third of ACL injuries resulting in post-traumatic osteoarthritis, or PTOA, within a decade.

Rat models have been extensively used to study the effect of ACL injury on PTOA, as the rat knee joint is a close model to the human knee joint. The most widely used model of ACL injury is ACL transection, where the joint is surgically destabilized. However, this model does not accurately replicate ACL injury conditions in humans.

In this video, we will discuss a novel, non-invasive rat ACL injury model, demonstrate the injury, and imaging of injured joint, and finally review research in the biomedical engineering field on ligament repair.

The knee consists of three bones, the femur, patella, and tibia. The anterior cruciate ligament, or ACL, is a band-like structure of dense connective tissue that ascends from the anterior intercondylar space of the tibia and extends superiorly and laterally to the posterior aspect of the lateral condyle of the femur.

The other ligaments in the knee include the posterior cruciate ligament, the lateral collateral ligament, and the medial collateral ligament. Structurally, all of the ligaments, especially the ACL, serve as passive stabilizers of the knee along with the thigh musculature to help control the joint during dynamic movement.

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. Animal models provide both a practical and clinically relevant way to study joint injury and treatment. The rat knee model in particular is widely used to study knee injury, as the rat knee closely resembles the human knee. To model a clinically-relevant ACL injury in humans, a single load of tibial compression is applied. When done correctly, this causes full rupture of the ACL.

ACL-injured hind limbs can then be imaged using micro-computed tomography, or Micro CT, to visualize joint injury and degeneration. Micro CT is an imaging technique that uses x-rays to create images of an object, like a joint. These cross-sections are measured across the object, and combined to create a three-dimensional reconstruction. For more information on micro CT, please watch the video in this collection.

Now that we've discussed the novel, non-invasive rat ACL injury model, let's take a look at how the injury is done, followed by micro CT visualization of the joint.

The ACL injury will be performed using a custom device, which will induce a single load of compression on the tibia of an anesthetized rat. First, place a rat in an induction chamber with five percent isoflurane and one liter per minute of oxygen. Once anesthetized, move the rat to the device using a nose cone to maintain a flow of one to three percent isoflurane. Position the right hind limb at 30 degrees of dorsaflexion and 100 degrees of knee flexion.

Move the top knee stage, which is mounted to a linear actuator, at one millimeter per second. Make sure to provide room for anterior subluxation of the tibia, relative to the femur. Then, position the flexed knee on the bottom stage, which is mounted directed above a load cell. Once the rat is properly positioned, turn the custom device on, open lab view, and input a compression speed of eight millimeters per second. Then, run the test to induce ACL rupture using a single load of tibial compression. As you run the test, monitor the procedure. The ACL injury is noted by the release of compressive force.

After injury, remove the rat from the device and place it on a flat surface. Then perform Lachman's test to assess the integrity of the ACL. While stabilizing the femur, pull the tibia forward. An intact ACL produces a firm endpoint, whereas an injured ACL produces a soft end feel. Once Lachman's test has been performed, return the rat to its housing to allow it to wake up from anesthesia.

Now let's image the damaged joint. To prepare for micro CT imaging, euthanize the rat in a humane way according to AVMA guidelines. Then extend and secure the ACL-injured hind limbs using several plastic zip ties, and carefully maneuver them into the custom device. The hind limb should be fully extended within the conical tube.

Secure the rest of the rat body in an appropriate container that is compatible with the micro CT stage. Then place the secured joint in the micro CT instrument and acquire two-dimensional images of the bones in the joint using scanner settings of 70 kilovolts at a current of 85.5 microangstroms and resolution of 11.5 microns for 180 degrees. Use an exposure time of five seconds at 0.6 degree rotation. Collect two-dimensional images, rotating every 0.6 degrees through the entire 180 degrees. Then reconstruct the images using an algorithm to create a three-dimensional image of the joint. To determine trabecular bone characteristics, first use a software plugin to acquire a volume rendering of the joint.

Then view orthogonal projections and move through slices to select the desired location between the epiphyseal plate of the medial and lateral tibial plateaus, and the medial and lateral condyles of the femur. Next, crop the knee at the desired location and mask it with a 1.53 millimeter sphere. Use interactive thresholding to label the bone and binarize the image. Now, compute the trabecular bone thickness, which is a measurement of the onset of osteoarthritis.

Repeat for different locations and to quantify other trabecular bone characteristics. After imaging, you may want to confirm ACL rupture by visual inspection and by opening up the knee. To do this, first remove the skin. You should see a hemarthrosis, which means there is blood in the capsule and is characteristic of an ACL injury.

Now, continue to open up the joint to expose the anterior distal femur, the patella, and the ACL. Perform a Lochman's test to open up the joint even further and observe blood in the joint and the isolated proximal tear of the ACL.

Now, let's compare the joint degeneration and trabecular bone structure in a rat knee with an acute ACL injury and a rat knee four weeks post-ACL injury. Here, we see reconstructed 3-D images of a rat knee with an acute ACL injury and at four weeks post ACL-injury. The trabecular bone thickness, number, and spacing is calculated at four different locations in the center of the epiphyseal plate and compared.

A smaller trabecular number, reduced trabecular thickness, and greater trabecular spacing, was evident four weeks after the noninvasive ACL tear, compared to the rat knee with an acute ACL injury. All of these are hallmark characteristics of the onset of post-traumatic osteoarthritis.

Various animal models are important not only for the study of the ACL injuries, but also to evaluate new treatments. One of the current treatments for ACL injury is the ligament reconstruction using a tissue graft. In this study, researchers created a fibrous tissue graft using polycaprolactone. The acellular graft was then implanted into rats, replacing the natural ligament.

The graft was secured to the knee joint by drilling holes in the femur and tibial plateau, and then passing the graft through the holes and securing with sutures. After 16 weeks, the histological analysis demonstrates that the scaffold matrix became infiltrated by fibroblasts and that the polymer was largely resorbed with little evidence of it remaining. Engineered ligaments can also be studied in vitro.

In this study, human cells were isolated from ACL remnants and expanded in culture. The cells were then cultured on coated plates with anchors to form engineered ligament constructs. After adding fibrinogen to encourage fibrin formation, the plates were cultured in an incubator.

After 28 days, the fibrin formed linear tissue between the two anchors. This type of study enables researchers to understand the role of different types of growth factors and hormones, to synthesize ACL replacement tissue, and determine ways to encourage ACL repair in vivo.

You've just watched Jove's introduction to the use of a rat model to induce and visualize ACL injury. You should now understand how the rat model is used to study and image ligament injury and several applications of this field of study.

Thanks for watching!