Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

We study tetrapod regeneration and use the axolotl as a top-tier regenerative species, comparing it to frogs and mice to discover cellular and molecular mechanisms of regeneration. The development of transgenics and genome editing, assembly of the axolotl genome, and application of RNA-Seq, ATAC-Seq, spatial transcriptomics, and proteomics allow us to deeper understand the regeneration microenvironment and compare it to less regenerative species, including humans. We established the cellular contribution to tissues in the regenerating limb and spinal cord, and major regulators of initial cell proliferation and positional identity in the blastema.

In a recent, paper we showed axolotl bone healing going through endochondral ossification, and the cellular level was similar to that of mammals. In contrast to previous techniques where the axolotl fracture was either unfixed or the neighboring bone served as a support, in the current protocol, the bone is fixated with a plate, thus, allowing to create a reproducible and aligned fracture and enabling a sound comparison to the studies in mice. The protocol allows for stable fracture with fixed cap size, broadening the studies on plate-fixated fractures to amphibians.

Despite their extreme regeneration abilities and full limb restoration upon amputations, axolotl surprisingly cannot heal large bone fractures with critical sized defects. We aim to define the pro-degenerative blastema factors to treat bone non-union in critical size defect. To begin, place the sedated Ambystoma mexicanum with the ventral side down on wet paper towels soaked in 0.03%benzocaine solution.

Cover the animal with benzocaine-soaked paper towels. Use a pair of ring forceps to stretch out the operative hind limb. With a scalpel, make a lateral longitudinal incision above the femur bone, spanning the whole thigh in the upper hind limb.

Carefully displace the muscles and nerves from the surgery site without cutting. Gently put Bode forceps under the femur to expose it for the surgery. Now place a rigid 7.75-millimeter four-hole fixator plate along the femur diaphysis without touching the joints and secure it in the aligned position with forceps.

Use four two-millimeter titanium screws to attach the bone to the plate. Under irrigation with 0.7x PBS and 1%penicillin streptomycin, manually drill to create the first hole in the bone for screw insertion. Then place the first screw in the hole.

Apply the saw-guiding device onto the first screw, and align it with the bone and plate. Use the saw-guiding device for drilling and inserting the remaining screws. After ensuring alignment of the plate with the bone, remove the saw guide, and use a screwdriver to break off the handles from the screws.

Place a piece of sterilized plastic film under the femur to prevent soft tissue damage during the osteotomy process. Then place a jiggly wire saw between the bone and protection film. Cut the bone using a jiggly wire saw, creating a single 0.7-millimeter cut in the femur.

Remove the saw and the protection film, and irrigate the surgery site with 0.7x PBS and 1%Penicillin-Streptomycin. Cover the top of the plate and screws with sterile bone wax to protect skin and muscles from irritation by the edges of the screws. Place muscles and skin on top of the wax.

Close the incision site with a size 7.0 synthetic suture, using simple interrupted stitches. After surgery, to reawaken, place the animal in a fresh artificial pond water tank, supplemented with Penicillin, Streptomycin, and Butorphanol. Micro CT and histological analysis facilitated the visualization of bone, fragment positions, and gap sizes, and the healing state.

Notably, Ambystoma mexicanum showed no callous formation three weeks post-surgery, contrasting with mammals. A cartilaginous callus was visible at three months, and an ossified bony callus appeared at six months post-surgery.