The overall goal of this procedure is to describe an experimental technique for the treatment of chondral defects in the rabbit knee joint. This is accomplished by first performing a cartilage biopsy from the knee joint to collect autologous chondrocytes. The second step is to extract, purify and expand the biopsied chondrocytes to a sufficient cell number in vitro.
Next, the chondrocytes are seated onto a three dimensional matrix. In the final step, the three dimensional matrix is implanted into a chondral defect that has been drilled into the contralateral knee of the rabbit. Ultimately, after 12 weeks in vivo, the implants can be addressed macroscopically with the repair tissue showing a homogeneous and intact surface of a solid quality.
This method can help answer key questions in the field of articular cartilage repair and may facilitate the experimental analysis of growth factor therapy, gene induction and changing metrics properties. After anesthetizing the rabbit shave the knee to be operated on with a pair of electric clippers. Then vacuum the fur and disinfect the shaved knee thoroughly cover the rest of the animal with a sterile dressing.
Next, palpate the patella. Perform a skin incision medially of the patella, and then open the knee joint by a medial para patella arthrotomy under sterile conditions, displace the patella laterally, and then use a sterile scalpel. To carefully peel small two to three millimeter long cartilage pieces out of the trochlea ossis femoris.
Immediately place the cartilage biopsies into a 50 milliliter tube containing complete medium, and then rinse the defects with sterile saline. Use forceps to remove any cartilage pieces that are left. Now reposition the patella within the trochlea groove and close the wound in layers with single button sutures and a continuing absorbable cutaneous suture.
Finally, seal the wound with a spray dressing that is permeable to water vapor. Begin this step by washing the sterile harvested cartilage twice in PBS with subsequent centrifugation for three minutes at 500 times G and room temperature. Next, cut the cartilage into one cubic millimeter pieces.
Transfer them to a new 50 milliliter conical tube, and then digest the pieces with 10 milliliters, tripsin, EDTA, and 10 milliliters PBS on a shaker. After 30 minutes, stop trypsin ization by adding 10 milliliters complete medium. After centrifugation, remove the supernatant and add serum free medium with collagenase A.Then shake the pieces for another 12 hours.
Now centrifuge the resulting solution, and then resuspend the cell pellet in five milliliters of complete medium seed the isolated cytes onto 25 square centimeter tissue culture flasks in five milliliters of complete medium and grow the cells in a humidified tissue culture incubator at 37 degrees Celsius 5%CO2. When the cultures have reached a density of 80%wash the cytes twice in PBS. Then expose them to 0.05%tripsin EDTA for three minutes.
After the cells have detached, add five milliliters of complete medium to stop the trypsin ization. After spinning down the cells, resuspend the pellet in complete medium, and then seed the cells in 75 square centimeter tissue culture flasks. After harvesting cells from an in vitro culture as just demonstrated, count and then transfer five times 10 to the fourth cells into a 1.5 milliliter micro centrifuge tube.
After spinning down the cell suspension, resuspend the pellet in 15 microliters of complete medium for a complete saturation of the matrix. Then cut a bilayer collagen scaffold to the exact dimensions of the trochlear defect and place the matrix into a 24 well plate. Now seed the chondrocytes onto the porous cell adhesive side of the matrix to detter cell migration away from the matrix.
Then incubate the cell seated matrices without further cell culture. Medium for one hour to allow full adherence of the chondrocytes. Finally, place the cell matrix constructs into a new 24 well plate with fresh, complete medium.
After performing a para patella arthrotomy to the contralateral knee as just demonstrated, begin the transplantation by using a sterile air operating power drill to create two isolated 3.6 millimeter diameter chondral defects in the trochlear groove. Next, rinse the defects with sterile saline. Taking care not to open the marrow cavity.
Now implant the chondrocyte seeded matrices with the porous side face down, and then use a little bit of fibrin glue to seal the implanted matrices to assure secure fixation. Relocate the patella within the trochlear groove and apply the full range of motion to the knee joint a few times. Then holding the matrices in place displace the patella laterally once again to check for any sign of fixation.
After replacing the patella, once again, finish the operation with wound closure in layers and spray dressing as just demonstrated. After 12 weeks in vivo, the chondral defect was filled by repair tissue with a homogeneous and intact surface, which reduced the shear stress and damage to the implant. Moreover, no hypertrophy or calcification of the implant was seen.
The repair tissue showed a stiff and solid quality, which was comparable to the healthy surrounding cartilage tissue. Moreover, a graft delamination did not occur. The red arrow indicates the pre-drilled chondral defect 12 weeks after seeding with the autologous chondrocytes.
After watching this video, you should have a good understanding of how to isolate autologous chondrocytes for subsequent proliferation in vitro and how to seed them onto a 3D metrics for reimplantation into artificially created cartilage defects in a rabbit knee. For further demonstration of the cartilage defect technique in a different experimental model, please also view our companion Jovi article, treatment of Osteocondral defects in the rabbit's knee joint by implantation of allogeneic mesenchymal stem cells in fibrin clots.
