This method enables investigation of the effects of different mechanical loading regimens, environmental conditions, or stages of cartilage degeneration on the vulnerability of in situ articular chondrocytes. The physical forces. Unlike other techniques, these measurements are performed in real time and in fully intact cartilage without compromising native boundary conditions.
Another advantage of this technique is it uses a mouse model, allowing testing of how specific genes affect the susceptibility of in situ chondrocytes in mechanical injury. For dissection of the distal femur, place the mouse in the supine position and make a five millimeter skin incision on the anterior portion of the knee. Extend the incision all the way around the knee.
And pull back the skin to expose the knee joint and leg muscles. Starting from the proximal end of the femur, use a number eleven scalpel blade to cut along the bone in the distal direction. Positioning the blade between the quadriceps muscle tendon unit and the anterior side of the femoral shaft.
Extend the incision past the patella. Cutting through the middle of the patellar tendon to remove the quadriceps muscle tendon unit. Next, starting from the proximal end of the femur, position the scalpel blade between the hamstring muscle tendon unit and the posterior side of the femoral shaft and cut along the bone in the distal direction.
When the incision approaches the knee joint, cut through the soft tissue, and finish the cut past the knee joint. Pull back the quadriceps and hamstring muscles to expose the femur. And cut away any excess muscle on the lateral and medial sides of the bone.
Cut the calf muscle on the posterior side of the proximal tibia. And flip the leg to visualize the posterior side of the femur. Remove the excess tissue around the knee joint to expose the distal condyles of the femur and the posterior surface of the proximal tibia.
Cut the anterior and posterior cruciate ligaments away from the femoral condyles. And pull the tibia away from the femur, cutting all the ligaments to separate the lower leg from the femur. Using standard dissection scissors, cut through the femur at the proximal end of the bone about eight millimeters above the tibiofemoral joint from the lateral side.
And use jeweler's forceps to remove the surrounding soft tissues from the femur. Then expose the cartilage on both condyles at the distal end of the femur. Periodically hydrate the articular surface with HBSS.
For dissection of the humerus, place the mouse in the supine position. And use micro scissors to make a five millimeter incision on the posterior side of the elbow. Extending the incision around the elbow and pulling back the skin to expose the muscles on the arm and shoulder.
Starting from the proximal end of the humerus, position the blade of a number eleven scalpel between the triceps muscle tendon unit and the posterior side of the humerus. And cut along the bone in the distal direction. Extend the incision toward the distal end of the humerus, through the triceps tendon and pull back the triceps toward the proximal end of the humerus until the humeral head is exposed.
Cut the connective tissue around the humeral head without touching the articular surface. And remove the limb from the body. Hydrate the articular surface of the humeral head with HBSS.
To disconnect the humerus from the arm, use forceps to break off the proximal end of the ulna on the posterior side of the arm. And cut the connective tissue around the distal end of the humerus, removing any excess tissue on the bone. Then use standard dissecting scissors to remove the deltoid tuberosity on the posterior side of the humerus.
And place the dissected specimen into a 1.5 milliliter microcentrifuge tube containing HBSS. For Calcein AM staining of the samples, transfer the bones into a 1.5 milliliter microcentrifuge tube containing 500 microliters of 10.05 micro molar Calcein in HBSS. And place the tube in a thermo mixer at 37 degrees Celsius at 800 rpm, protected from light.
After thirty minutes, wash the specimen in fresh HBSS for ten minutes. And transfer the specimen onto the glass slide of a custom microscope mounted mechanical testing device, such that the articular surface of the posterior femoral condyles or the humeral head is sitting on the glass. Hydrate the specimen with HBSS and place the device with the specimen onto a fluorescence microscope stage.
Image the articular chondrocytes stained with Calcein AM before the load application under a 4x magnification. Adjust the acquisition settings to optimize the image quality. To apply static mechanical loading, hold a prescribed static load on top of the specimen such that the articular cartilage is compressed against the cover glass for five minutes before removing the load.
To apply an impact mechanical load, drop a cylindrical impactor of known weight onto the specimen from a prescribed height, releasing the load five seconds after the impact. Immediately after either load has been removed, incubate the specimen in freshly prepared 60 micro molar propridium iodide in one milliliter of HBSS. For five minutes at room temperature, then re-image the articular chondrocytes by fluorescence microscopy as just demonstrated.
To quantify the area of injured and dead cells due to the applied mechanical loading regimen, first open the micrographs of the articular surface acquired before and after application of mechanical load in ImageJ. Combine the images into a stack. Set the scale of the images based on the image resolution.
Next, define the area where the cells became Calcein negative and PI positive. These cells are considered to be injured or dead. Then determine the area of injured dead cells using the measurement tool.
At least six tested applied the loading protocols reproducibly induced quantifiable localized areas of cell injury in femoral and humeral cartilages obtained from eight to ten week old valve C mice. In all of the loading protocols tested, higher load magnitudes and higher impact intensities significantly exacerbated the spacial extent of cell injury in both femurs and humeri. While performing the dissections it is important to remember not to contact the articular cartilage with the surgical tools, as this may compromise the baseline by ability of the specimens.
The use of this mirroring model may facilitate research in osteoarthritis, as the disease is easily induced in mice through genetic, dietary, or surgical manipulations. Our platform also provides a tool for interrogating basic science questions and for screening therapeutic interventions targeting the mechanical vulnerability of chondrocytes.
