All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Dissection of the bovine intervertebral disc
- Rinse the whole tail thoroughly with tap water to remove dirt and hair on the surface.
NOTE: With intact, distal ends, a maximum of 9 intervertebral discs (IVDs) (coccygeal 1-9) per tail can be used for the experiments depending on the desired size of the IVDs. Considering the desired diameter between 15-20 mm, we used 12 bovine tails with 5 IVDs per tail for the experiments.
- Immerse the whole tail in a box containing 1% betadine solution for 10 min. Briefly dry the tail with sterile gauze and place it on a sterile drape.
NOTE: During dissection of the disc, humidify the tails with Ringer's solution wetted gauze to prevent dehydration. Store the tails (or left-over segments) wrapped in wet gauze until the whole dissection procedure is completed.
- Use a scalpel (No. 20) to remove the soft tissue as completely as possible from the caudal spine to facilitate the identification of the IVDs. Remove the spinous and transverse processes of the vertebrae with bone removal pliers.
NOTE: Select IVDs with the desired diameter. IVDs with a diameter range of 15-20 mm were used in the current study.
- Cut transversely with bone pliers through the middle of each vertebral body to obtain individual motion segments. Put motion segments in a Petri dish with gauze wetted with Ringer's solution.
- Locate the IVD and vertebra by palpation and by moving the motion segments gently. Make two parallel cuts with the band saw in the growth plate of the IVDs, one on each side of the IVD. Identify the location of the growth plate by touching and finding the convex site of the bony endplate part (hard) adjacent to the disc (soft) with a safety distance of approximately 0.5-1 mm from the IVD towards the vertebra. Ensure that the blade of the band saw is cooled with Ringer's solution while cutting the vertebrae.
- Transfer IVDs in a clean Petri dish with clean gauze wetted with Ringer's solution.
NOTE: The gauze should be moistened and not too wet to prevent swelling of the IVDs,
- Use the scalpel blade to scrape off the vertebral body (red/pink bone), growth plate (white cartilage), leave the endplate intact (yellow-pink). Make the two surfaces flat and parallel for the loading procedure. Transfer scraped IVDs to a fresh Petri dish with gauze wetted with Ringer's solution.
NOTE: Wear a chainmail glove to protect the hand while holding the IVD and scraping.
- Measure the disc height and diameter with a caliper. Clean the blood clots in the vertebrae bone with Ringer's solution using a jet lavage system.
- Transfer the IVDs to 50 mL plastic tubes, one IVD per tube. Add 25 mL of Phosphate-Buffered Saline (PBS) + 10% Penicillin/Streptomycin (P/S) per IVD and leave it shaking for 15 min on an orbital shaker at room temperature.
- Aspirate the supernatant and add 10 mL of PBS + 1% P/S per IVD for 2 min to rinse the IVDs.
2. IVD culture and loading
- Transfer discs to IVD chambers and add IVD culture medium (Dulbecco's Modified Eagle Medium (DMEM, 4.5 g/L high glucose DMEM for the physiological group and 2 g/L low glucose DMEM for the pathological group) + 1% P/S + 2% fetal calf serum + 1% ITS (contains 5 µg/mL insulin, 6 µg/mL transferrin, and 5 ng/mL selenious acid) + 50 µg/mL ascorbate-2-phosphate + 1% non-essential amino acid + 50 µg/mL antimicrobial agent for primary cells) and place in an incubator at 37 °C, 85% humidity and 5% CO2.
- Culture the discs for 4 days within a bioreactor system according to experimental groups. In the pathologic group, maintain degenerative loading conditions at 0.32-0.5 MPa, 5 Hz for 2 h/day. In the physiological control group, use a loading protocol of 0.02-0.2 MPa, 0.2 Hz for 2 h/day.
NOTE: Position the IVDs in chambers containing 5 mL of IVD medium during the loading procedures. The volume depends on the size of the bioreactor's loading chambers. Between the loading procedures, place the IVDs in six-well plates with 7 mL of IVD culture medium for free-swelling recovery.
- For analyzing the changes in disc height during the experimental period, measure the disc height with a caliper after IVD dissection (baseline) and then daily after the free swelling period and after dynamic loading for the experimental duration.
