In Vitro Impact Model to Generate Sublethal Chondrocyte Injury in Bovine Cartilage Explants

High mechanical loads to cartilage, such as those in joint injuries, can lead to post-traumatic osteoarthritis. However, the molecular pathways that drive this process are not fully understood.

Mechanical loads can be applied to cartilage explants using a drop tower or other device. But loads that are too high or not uniform can kill the chondrocytes.

We developed techniques to harvest bovine cartilage explants, and apply a controlled load using a drop tower to injured chondrocytes without causing immediate death.

These protocols allow us to study the early injury response in viable cells that are potentially treatable.

Our ultimate goal is to uncover molecular pathways that are triggered by chondrocyte injury, and to find ways to stop disease progression before it begins.

[Instructor] To begin, obtain bovine forelegs from skeletally mature animals at a local abattoir within six hours of sacrifice. Keep the tissue on ice during transportation. Using a meat band saw, remove the hoof to isolate the metacarpophalangeal joint without opening the joint capsule. With a number 22 scalpel blade, remove the skin carefully without puncturing the joint capsule. Rinse each leg thoroughly outside the bio safety cabinet with cold water to remove dirt. Then apply Betadine to coat and disinfect the surface and rinse. Now, inside the tissue culture hood, use a new number 22 scalpel blade to open the joint capsule. Cover the articular cartilage with sterile gauze soaked in PBS to maintain hydration, then isolate the distal joint. To isolate the distal metacarpophalangeal joint, cut through the metatarsus with an autopsy saw. Place the distal joint into a vice with the covered cartilage facing upwards, and tighten the vice until secure. Regularly moisten the gauze on the cartilage with PBS throughout the process. Now, fold the gauze away from one condyle. And adjust the position of the vice so that the coring bit is aligned above the flattest region of the articular surface. Then open the carboy valve to allow PBS to flow through the coring bit. To core into the condyle slowly, press the drill bit into the condyle surface while applying PBS from a wash bottle. Frequently release pressure to allow heat to dissipate, while continuing coring to a depth of approximately two millimeters into the subchondral bone. Next, use the autopsy saw to release osteochondral cores and cut parallel to the articular cartilage surface while applying PBS. Store the osteochondral cores in dilute Betadine solution until all joints have been processed. Prepare a 12 well plate with three milliliters of prewarmed culture medium in each well. Then rinse osteochondral cores in PBS, and transfer each sample into an individual well of the six well plate. Place the samples in a humidified incubator at 37 degrees Celsius with 5% carbon dioxide until the impact procedure on the next day. Fix the impact holder to the base plate of the drop tower using four autoclave screws. Place an osteochondral core into the holder under the impactor head of the drop tower with the bone side facing upwards. And adjust the drop tower carriage to the desired height above the bone surface. Click on the start button on the data acquisition software just before releasing the spindle stop. This frees the drop tower carriage to drop by gravity onto the sample. Immediately lift the carriage and rest it back on the spindle stop. Now, load the text file generated and run the code to filter raw data and calculate impact parameters. Finally, using a number 11 scalpel blade, remove cartilage from the bone. Return the cartilage sample to the culture media, and place it back in a humidified incubator at 37 degrees Celsius with 5% carbon dioxide until imaging the next day. The mechanical impact load to osteochondral explants was applied using a drop tower with different carriage heights. Increasing the drop carriage height from four to five centimeters resulted in a stepwise increase in peak load, mean peak stress, loading rate, and impact energy. Cartilage impacted at five centimeters showed a significantly lower percentage of viable cells 24 hours after impact compared to non-impacted samples. The intensity of apoptotic florescence increased significantly with impact from four centimeters, and rose further at 4.5 and five centimeters. The fitted holder with a diameter only 0.1 millimeter larger than the sample preserved significantly higher cell viability after impact compared to the loose holder. Apoptotic signaling was more concentrated at the surface, and significantly higher in samples impacted with a loose holder compared to a fitted one. Removing the cartilage from the bone immediately resulted in significantly higher intensity of fluorescent staining for apoptotic signaling.