Generating a Pro-Inflammatory Organ Culture Model to Simulate an Early Intervertebral Disc Disease
Transcript
Start with rinsing the entire bovine tail thoroughly with tap water to remove dirt and hair on the surface. Then, immerse the tail in 1% betadine solution for 10 minutes to disinfect the surface. Use a scalpel number 20 to remove the soft tissue from the caudal spine to facilitate the identification of the intervertebral discs or IVDs.
Remove the spinous and transverse processes of the vertebrae with a bone removal plier. Cut transversely with bone pliers through the middle of each vertebral body to obtain individual motion segments, then put the collected motion segments in a Petri dish with a gauze wetted in Ringer's solution.
Locate the IVD and vertebrae by moving the motion segments gently. Then, identify the location of the growth plate by touching and finding the convex side of the bony end plate. Cool the blade of the bandsaw with Ringer's solution, and use it to make two parallel cuts in the growth plate of the IVDs, one on each side.
Transfer the IVDs to a clean Petri dish with clean gauze wetted with Ringer's solution. Scrape off the vertebral body and growth plate using the scalpel blade, leaving the end plate intact. Position the two surfaces flat and parallel for the loading procedure, and transfer scraped IVDs to a fresh Petri dish with gauze wetted with Ringer's solution.
Measure the disc height and diameter with a caliper. Then, clean the blood clots in the vertebrae bone with Ringer's solution using a jet lavage system. Disinfect IVDs in PBS and 10% penicillin-streptomycin, with shaking for 15 minutes. Rinse off the high-concentration antibiotics with PBS and 1% penicillin streptomycin.
Transfer discs to IVD chambers containing 5 milliliters of IVD culture medium, and place them in the bioreactor system with an incubator at 37 degrees Celsius with 85% humidity and 5% carbon dioxide. Culture the discs for four days within a bioreactor system by maintaining different loading conditions according to the experimental groups.
In the physiological control group, culture the IVDs with high glucose medium using a loading protocol of 0.02 to 0.2 megapascals and 0.2 Hertz for two hours per day. In the pathological group, culture the IVDs with low glucose medium using a loading protocol of 0.32 to 0.5 megapascals and 5 Hertz for two hours per day.
Between the loading procedures, place the IVDs in six-well plates with 7 milliliters of IVD culture medium for free swelling recovery. Measure the disc height daily with a caliper after the free swelling period and after dynamic loading for the experimental duration.
