The protocol aims to simulate in vivo conditions of intervertebral disc degeneration to investigate its pathophysiology without the need for animal models. Compared with in vitro cell culture, the bioreactor organ culture technique maintains the cells in their native biological and biomechanical microenvironment in addition to the supply of a controllable and reproducible culture condition. 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 in 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 five 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 disks 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 five hertz for two hours per day.
Between the loading procedures, place the IVDs in six-well plates with seven 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. After the first dynamic loading cycle on day one, directly place the IVDs in a Petri dish in a vertical position and stabilize them with a tweezer.
Inject recombinant TNF-alpha with a 30-gauge insulin needle slowly at a speed of approximately 70 microliters per minute into the IVDs of the pathological group. After injecting, pull the syringe halfway back and pull the plunger to create a vacuum that prevents the injected solution from leaking, then remove the syringe completely from the IVD. Degenerative loading combined with limited nutrition supply and a TNF-alpha injection caused a significant increase in the gene expression of pro-inflammatory markers interleukin six and interleukin eight in NP tissue after four days of culture.
The interleukin eight protein release into the conditioned medium showed a marked increase in the pathological group on day two and day four. Disc height reduction after loading was higher in the pathological group compared to the physiological group, and the differences were more pronounced after days two and three compared with day one, indicating a progressive effect of the degenerative and inflammatory conditions. A standardized dissection technique is important for reproducible IVD whole-organ culture models.
A bioreactor is required to apply physiological and degenerative loadings on IVDs to simulate various in vivo biomechanical conditions. This technique can be applied on human IVD explants which is of high clinical relevance. It's a novel preclinical model for developing effective treatments on early stage disc degeneration.
