The overall goal of this procedure is to demonstrate a standardized method for experimental stem cell research in the field of osteochondral repair. This is accomplished by first isolating mesenchymal stem cells from rabbit bone marrow. In the second step, the isolated stem cells are cultured to facilitate proliferation.
Next, the fibrin cell clots are manufactured in vitro. Finally, the pre-established fibrin cell clots are implanted into artificial osteochondral defects in the rabbit knee joint. Ultimately, after 12 weeks in vivo, a macroscopic analysis of the knee can be performed with the repair tissue showing a homogeneous and intact surface with a solid texture.
The implications of this technique extend toward the therapy of cartilage lesions in an experimental animal setting as it allows for in vivo analysis of the integration and regeneration capacity of allogeneic mesenchymal stem cells for the treatment of cartilage damage. In the surgery room, use a pair of electric clippers to shave the fur from the hind limbs back and belly of a euthanized rabbit, and simultaneously vacuum the fur after disinfecting the shaved area thoroughly with 70%ethanol. Make an incision along the cranial surface of the leg and calf, and then reflect the skin and subcutaneous tissue by sharp dissection.
Next, separate the muscles and ligaments from the tibia and femur, keeping the cuts as close to the bone as possible for a clean dissection. Then cut through the hip joint to separate the head of the femur from the acetabulum and elevate the tibial femoral complex. Now use the scalpel blade to scrape off any remaining soft tissue from the bones.
Then cut away the patella and the knee joint ligaments to finally separate the leg bones. Spray the separated bones with 70%ethanol after they have air dried. Place each bone into separate 50 milliliter centrifuge tubes containing cell culture media to keep them moist in a sterile cell culture.
Laminar flow hood. Use sterile forceps to transfer the harvested bones from the centrifuge tubes into 150 millimeter dishes. Then use a sterile saw to remove both of the bone ends and move the pieces to new 150 millimeter dishes.
Next, fill a 10 milliliter syringe with medium. Attach an 18 gauge needle to it and insert the tip of the needle into one end of the bone into the marrow. Now rinse the marrow cavity with medium to flush the bone marrow into the dish.
Then aspirate the cell suspension into the syringe and rinse the bone marrow repeatedly until the suspension is free, floating through the bone marrow cavity and no further bone marrow clots appear. Once the bone marrow has been collected from all the bones, fill the syringe with the bone marrow suspension through the needle and force the marrow suspension out into medium to disrupt the marrow clumps. Afterwards, pass the suspension through a cell filter into a 50 milliliter tube to prevent cell loss.
Wash the culture dish twice with 10 milliliters of medium, pouring the wash through the filter each time as well. Centrifuge the suspension for five minutes at 500 times GE and room temperature. Then remove the supernatant and resuspend the cell pellet in 10 milliliters of medium.
Next, add five milliliters of bio call separating solution into a 15 milliliter tube, and then carefully layer five milliliters of cell suspension on top. Then after centrifuging the layered solution, the red blood cells will sediment to the bottom while the PBMC and mesenchymal stem cells will remain at the interface. Transfer the cells at the interface to a 15 milliliter conical tube and use five milliliters of PBS to wash the collected PBMC and mesenchymal stem cells two to three times.
Then spin the cells one more time and resuspend the pellet in 10 milliliters of medium. Finally, count the cells with a hemo cytometer and then plate them at an initial seeding density of approximately five times 10 to the sixth in 150 millimeter dishes. After two to three days, maintain the cultures by removing the non-adherent cells.
Rinsing the plate with PBS to remove the cell debris and feeding the cells with fresh, complete medium on the day of implantation. Begin by exposing the adherent cells to 0.25%trypsin EDTA for three minutes. Stop trypsin ization by adding complete medium.
Then transfer the detached mesenchymal stem cells to a 50 milliliter conical tube and wash them twice with PBS. After determining the cell count and viability by trian blue staining, prepare a master mix of at least one more clot as needed for n equals four. Spin down 200, 000 cells in a micro centrifuge tube and then resuspend the pellet in 68 microliters of PBS and 100 microliters of the fibrinogen component of a tissue coal kit.
Next, inoculate four microliters of thrombin solution into one hole of a pre-drilled sterile plate. Immediately add all 42 microliters of one fibrinogen cell suspension, and then top the cell suspension with four more microliters of thrombin solution. Initially, the 50 microliter volume of the pipetted fibrinogen cell suspension, we'll protrude over the rim of the pre-drilled hole due to the surface tension.
However, after an hour, the admixture will be completely clotted and will have contracted to fit into the pre-drilled hole. Now, use blunt forceps to carefully remove the clot and then place it into a micro centrifuge tube containing PBS. After disinfecting the shaved knee, thoroughly palpate the patella and perform a skin incision medial to the patella.
Next, open the knee joint by a medial para patella arthrotomy under sterile conditions, and then displace the patella laterally. Now use a sterile air operating power drill with a 3.6 millimeter in diameter drill bit and a stop device to create two three millimeter deep figure eight shaped osteochondral defects in the trochlea groove. Then after rinsing the defects with sterile saline, dispense 20 microliters of fibrin glue evenly into the bottom of the defects quickly before the glue dries.
Implant the press fitted clots into the figure eight shaped defect. After clotting, relocate the patella within the trochlear groove and bend and stretch the knee a few times. Then displace the patella laterally once again and check if the fibrinogen cell clots are still in place.
After replacing the patella again, close the wound in layers with single button four aut Vicryl sutures and a continuous four aut Monocryl cutaneous. Finally seal the wound with a spray dressing permeable to water vapor. In this experiment, the defect was filled by repair tissue with similar biomechanical properties and similar durability as the surrounding cartilage.
This fibrin cell clot was prepared in vitro on a sterile plate with pre-drilled holes, which had the same size as the osteocondral defect. As a result, there were no clefts between the implanted fibrin clot and the surrounding cartilage, which would be a risk factor for premature degeneration or delamination. A basal hailing of the repair tissue was ensured because the subc chondral bone was penetrated and thus worked against a shearing.
Another important aspect was the stiffness of the repair tissue, which should match the healthy surrounding cartilage tissue to avoid an increased load on the joint and a possible premature degeneration. Moreover, an intact and homogeneous surface of the transplant was found, which reduced the sheer stress and possible implant damage After its development. This technique could pave the way for researchers in the field of orthopedic stem cell research to explore treatment options for osteochondral defects and an experimental animal model.
For more explicit demonstration in some of the animal preparation and knee surgery techniques, be sure to view our companion Jovi article matrix assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects. In a rabbit model.
