Overview
Source: Lindsey K. Lepley1,2, Steven M. Davi1, Timothy A. Butterfield3,4 and Sina Shahbazmohamadi5,
1Department of Kinesiology, University of Connecticut, Storrs, CT; 2Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT; 3Department of Rehabilitation Sciences, University of Kentucky, Lexington, KY; 4Center for Muscle Biology, Department of Physiology, University of Kentucky, Lexington, KY; 5Biomedical Engineering Department, University of Connecticut, Storrs, CT
Anterior cruciate ligament (ACL) injury to the knee dramatically increases the risk of post-traumatic osteoarthritis (PTOA), as approximately one-third of individuals will demonstrate radiographic PTOA within the first decade following ACL injury. Though ACL reconstruction (ACLR) successfully restores knee joint stability, ACLR and current rehabilitation techniques do not prevent the onset of PTOA. Therefore, ACL injury represents the ideal model to study the development of PTOA after traumatic joint injury.
Rat models have been used extensively to study the onset and effect of ACL injury on PTOA. The most widely used model of ACL injury is ACL transection, which is an acute model that surgically destabilizes the joint. Though practical, this model does not faithfully mimic human ACL injury due to the invasive and non-physiological injury procedures that mask the native biological response to 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.
Visualization of joint degeneration through micro-computed tomography (µCT) provides several major advancements over traditional OA staining techniques, including rapid, high-resolution, non-destructive 3D imaging of whole joint degeneration. The goal of this demonstration is to introduce the state of the art non-invasive ACL injury in a rodent model and use µCT to quantify knee joint degeneration.
Principles
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.
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Procedure
Non-invasive ACL Injury
- Wear proper personal protection equipment. You may use a respiratory mask, but it is not mandatory for this protocol.
- Anesthetize the rats using an induction chamber with 5% isoflurane and 1 L/min oxygen. Maintain the flow of anesthesia using via a nose cone with 1 - 3% isoflurane and 500 mL/min of oxygen. If the apparatus is not set up on a backdraft or downdraft table, ensure that waste gas is scavenged using a tabletop system and charcoal filters.
- Perform a toe-pinch to ensure an adequate depth of anesthetic has been reached. Note that it is not necessary to apply eye lubricant the protocol is performed quickly (< 3 min) and there is minimal risk of cornea dryness.
- Position the right hindlimb at 30° of dorsiflexion and 100° of knee flexion while providing room for anterior subluxation of the tibia relative to the femur.
- Rigidly mount the top knee stage to a linear actuator.
- Position the flexed knee on the bottom stage, which is mounted directly above a load cell.
- Induce ACL injury using a single load of tibial compression at a speed of 8 mm/s.
- ACL injury is noted by a release of compressive force. This is monitored via a custom program.
- Post-injury, while the animal is still under the plane of anesthesia, perform a Lachman's test to clinically confirm an ACL rupture has occurred. A Lachman's test is a clinical test used to evaluate the integrity of the ACL by assessing sagittal plan stability. While stabilizing the femur, pull the tibia forward (in an anterior direction) to assess the amount of motion. An intact ACL will produce a 'firm end-feel' where the researcher will not be able to translate the tibia forward. An injured ACL will produce a 'soft or mushy end-feel,' indicative of a torn ACL.
- Palpate the femur and tibia to detect any gross bone damage. If no contraindications are identified, transfer the animal to its cage and allow it to recover. During this time, monitor the animal to ensure it doesn't display any signs of pain, such as a reluctance to move, vocalization, or abnormal posturing.
µCT imaging of joint degeneration
2-D images are obtained using scanner settings of 70 kV, current 85.5 µA (Figure 2B). Data is collected every 0.6° rotation step at a resolution of 11.5 µm through a complete 180°. Cross-sectional images are reconstructed using a smoothed back-projection algorithm and on the stack of reconstructed images (Figure 2C). Trabecular structure is then analyzed by segmentation in software, whereby a 1.53 mm sphere is centered in the epiphyseal plate of the medial and lateral tibial plateaus and femur to determine trabecular thickness (µm), trabecular separation (µm) and trabecular number (1/mm).5,6
- At 4 weeks post-ACL injury, euthanize the rat with prolonged exposure to CO2 in the induction chamber.
- Extend and secure ACL injured hindlimb in a custom 3D-printed device (Figure 2A).
- Acquire images using µCT.
- Obtain frontal plane radiographs to determine joint space. narrowing (between the femoral condyle and tibial plateau [measured in mm]) compared to the non-injured limb.
- Obtain 2-D images using the following scanner settings: 70 kV and current 85.5 µA.
- Collect the data every 0.6° rotation step at a pixel size of 11.5 µm through a complete 180°.
- Reconstruct cross-sectional images using a smoothed back-projection algorithm on the stack of reconstructed images.
- To ensure a consistent region of interest is measured, place a 1.53 mm sphere in the epiphyseal plate of the medial and lateral tibial plateaus and femur to determine trabecular thickness (µm), trabecular separation (µm), and trabecular number (1/mm).
Figure 2: A) Custom printed device to hold hindlimb during μCT, B) 2-D images, and C) 3-D μCT.
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.
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Results
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.
| Animal | Tb.N (1/mm) |
Tb.Th (µm) |
TB.Sp (µm) |
| Acute ACL injured | 3.11 | 168.5 | 217 |
| 4 wks post-ACL injury | 2.63 | 166.7 | 213 |
Figure 4: Image of an acute injured ACL-limb (left) and image of intact, healthy ACL (right).
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Applications and Summary
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.
Materials List:
| Equipment | Company | Catalog Number | Comments |
| Linear actuator | Phidgets | L16-63-12-P | |
| Load Cell | HDM Inc. | PW6D | |
| μCT | Zeiss | XRM Xradia 520 |
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References
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- Lockwood KA, Chu BT, Anderson MJ, Haudenschild DR, Christiansen BA. Comparison of loading rate-dependent injury modes in a murine model of post-traumatic osteoarthritis. J Orthop Res. 2014;32(1):79-88.
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- Mohan G, Perilli E, Kuliwaba JS, Humphries JM, Parkinson IH, Fazzalari NL. Application of in vivo micro-computed tomography in the temporal characterisation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate-induced osteoarthritis. Arthritis Res Ther. 2011;13(6):R210.
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