The overall goal of this surgical intervention is to create a femoral critical-size bone defect stabilized by plate osteosynthesis in mice. This method can help answer key questions in the bone tissue engineering field about the bone-healing capabilities of specific bone substitute of the process used involved in bone regeneration. The main advantage of this technique is that it establish a reproducible bone defect in a load-bearing bone pertinent to orthopedic application of the model.
After aseptic limb scrubbing, a sterile plastic transparent drape covering the mouse is placed, and a small hole in the drape is made to isolate the leg. With the anesthetized mouse in ventral recumbency, and the left hind limb in extension, make a 15 to 17 millimeter longitudinal skin incision over the anteriolateral aspect of the femur, extending from the hip joint to the stifle joint. Next, incise the fascia lata and split the vastus lateralis and biceps femoris muscles to expose the full length of the femoral diaphasis.
To enhance the femoral diaphasis exposure, transect the gluteal and biceps femoris muscles from the third trochantor and circularly dissect the femur at the middle of the diaphasis. Then, apply a six-hole titanium micro-locking plate to the anterior femoral side of the bone. And use a zero point three millimeter drill bit to bore the most proximal hole of the plate.
Use a dedicated screwdriver to insert the first screw while holding the plate parallel to the femur. Then, holding the plate in position, drill the most distal hole of the plate and insert and lock the second screw. Insert the two final screws without locking.
Now, place the wire of a zero point two millimeter jiggly saw closely around the femur in a mediolateral orientation. Insert the dedicated jig onto the stems of the last two screws, and apply the jig above the plate. Then, under sterile isotonic saline irrigation, make a three point five millimeter long mid-diaphasial femoralistectomy using the middle two thirds of the wire while applying a constant steady tension against the bone.
When the bone has been severed, cut the saw wire close to the bone on one side and remove the jig. Lock the last two screws, then copiously rinse the surgical field with sterile isotonic saline. Afterwards, surgically fill the segmental defect with the test material of interest, and position the vastus lateralis muscle loosely over the plate.
Finally, use five-zero glycomer 631 sutures in a simple continuous pattern to close the fascia and subcutaneous planes, closing the skin with four-zero glycomer 631 sutures and a simple interrupted suture pattern. The weight of the osteosynthesis used in the present study was about zero point one percent of the mouse's bodyweight and no post-operative complications, self-injury, or cage-mate inflicted injuries occurred. Functional recovery of the operated limb was excellent in all of the animals and full weight-bearing was observed within a day after surgery.
When the surgically induced bone defects were left empty, no significant bone formation was observed. With a consistent bone non-union in all animals. In contrast, when the defects were filled with either an isograft or a coral scaffold, newly formed bone extending from the proximal and distal bone edges was observed.
However, whereas bone formation allowed the re-establishment of bone continuity, in most of the defects treated with isografts, bone re-establishment was observed only between the scaffold and the bony edges of the coral-filled defects. Bone union did not occur in any animal of the defect-empty group ten weeks post-implantation. In the isograft and coral scaffold groups, bone union was achieved in most of the animals with very few bones observed inside the scaffold itself.
Once mastered, this technique can be completed in 45 to 60 minutes if it is performed properly. While attempting this procedure, it is important to remember that the bone stability is achieved by the plate osteosynthesis, and therefore the plate should be applied in the correct position onto the femoral bone. Following this procedure, other method like micro-CT and bioluminescence imaging can be performed to answer an additional question about the bone formation and implantants at survival when a self-seeded scale form is used as a defect filler.
After its development this technique paved the way for researcher in the field of bone tissue engineering to explore bone regeneration processes. After watching this video, you should have a good understanding of how to generate a reproducible bone defect in a murine load-bearing bone that is stabilized by plate osteosynthesis. Don't forget that working with animals and surgical implements can be extremely hazardous and that precaution such as maintaining sterility, careful anesthesia usage, and attention to animal welfare should always be taken while performing this procedure.
