This method models many aspects of human post-traumatic osteoarthritis, allowing us a way to investigate the disease and the impact of possible therapies in a shorter timeframe. The main advantage of this technique is that in the short-term, just two weeks after intervention, the mice displayed consistent, measurable osteophyte formation, as well as changes to load in the affected leg that indicate pain. We recommend using this model of the study of osteophyte formation and endochondral ossification, as well as injury-driven pain.
The identification and cutting of the medial meniscotibial ligament can be challenging, so we advise practicing this on cadavers until confident. Helping to demonstrate the procedure will be Lynette Dunning, a research assistant in the Centre for Musculoskeletal Science. To begin, designate a sterile room to carry out the surgery, ensuring that all surfaces are sterile.
Arrange and place sterile instruments on sterile drapes. Then, weigh the mouse. Clip the fur of the anesthetized mouse over the knee, front and lateral sides from the mid-shin to the mid-thigh, with small hair clippers.
Disinfect skin by applying antibacterial skin cleanser on shaved, exposed skin. For analgesia, administer 0.05 milligrams per kilogram of buprenorphine subcutaneously. Place the mouse on the dorsal side, leaving the knee on which to be operated upwards, and place the mouse's nose in the nozzle connected to the anesthetic rig.
Cover the mouse with a sterile drape with a small keyhole opening. Position the leg on which to be operated with the knee fixed at less than a 90-degree angle, with the patellar ligament facing upwards and the foot immobilized with surgical tape. Adjust the microscope to focus on the patellar ligament.
Pinch the skin of the knee on the lateral side with serrated forceps. Make a small cut parallel to the distal patellar tendon using surgical scissors. Introduce the scissors and expand the cut to about 10 millimeters.
Move the skin over to the medial side, exposing the patellar ligament and the proximal tibial plateau. Using a number 11 blade, make an incision from top to bottom along the medial side of the patellar ligament. When reaching the bottom of the patellar ligament, turn the blade 90 degrees and extend the incision away from the patellar ligament toward the medial side, to gain access to the joint capsule.
Pinch the patellar ligament with blunt-tip forceps and rotate the wrist to move the patellar ligament to the lateral side, just enough to expose the IFP. While still holding the patellar ligament lightly, pinch the IFP with micro tweezers to raise it and move it slightly upward. If there is a bleed, apply pressure with a cotton bud.
Identify the MMTL of the medial meniscus, which anchors the cranial horn of the medial meniscus to the anterior tibial plateau. Carefully sever the MMTL with small, two-millimeter blade spring scissors, leaving the medial meniscus and other ligaments intact. With a three millimeter microsurgical knife, mark three, evenly-spaced indentations on the tibial articular cartilage in a direction from the posterior to the anterior part.
Close the skin with two or three small, seven-millimeter wound closure metal clips. Remove metal clips between five to seven days post-surgery. Then, evaluate pain or gait at any point during the study.
Quantify calcified tissue by analyzing the Micro-CT scans of the knee joints. To analyze subchondral bone sclerosis, select a VOI in the center of the medial tibial plateau load. Then, determine the subchondral bone density and micro architecture by selecting a region of interest delineating the trabecular structure with the tibial epiphysis, the subchondral plate, or the total subchondral bone in the two-dimensional coronal view of the stack, using a CT analyzer software.
Identify osteophytes in the reconstructed, three-dimensional image stacks using CTVol software. Evaluate cartilage damage and synovitis according to the OARSI cartilage damage score 19 and synovitis score 20, on paraffin-embedded, five-micrometer sections. There were no significant changes in the rear leg load in the DMM model within eight weeks post induction, while DCS mice favored the contralateral, or control leg, significantly, two weeks after intervention.
The ratio between the contralateral and the ipsilateral leg indicated that both models had increased bone density in the subchondral bone loading area of the affected limb, four weeks after induction. The emergence of osteophytes was more prominent in the DCS mice, with a significant increase in the number and volume compared to the DMM model two weeks after intervention. DCS presents elevated cartilage damage in the medial tibial and femoral compartments, and synovitis four weeks after induction.
Damage should be limited to the structures we intentionally want to affect, so special care is needed to maintain all other structures intact and minimize the exposure of the cartilage. The modification to include intentional cartilage scratches allows us to investigate osteoarthritis with a focus on osteophytogenesis, early osteoarthritis, or injury pain, and the effect of cartilage damage on the whole joint.
