In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The overall goal of this technique is to use magnetic resonant imaging, or MRI, for the longitudinal evaluation of murine fracture callus development during bone healing. This method can help answer key questions in the bone healing field such as how fracture calluses develop over time in different mouse models. The main advantages of this technique are that fracture callus development can be monitored in vivo in real-time and that the applied external fixator provides a highly standardized fracture healing model.

Demonstrating the MRI procedure will be Anne Subgang, a technician from the Small Animal Imaging Core facility. Before beginning the procedure, confirm the appropriate level of sedation by lack of response to toe pinch and inject the mouse subcutaneously with a single dose of antibiotics in a fluid depot of two times 250 microliters of 0.9%sodium chloride. Apply ointment to the animal’s eyes and place the mouse on a 37-degrees-Celsius heating pad.

Remove the fur from the right hind limb and scrub the surgical area with an alcohol-based disinfectant. Place a sterile drape over the animal leaving the surgical area exposed, and cover the right hind paw with a small piece from a sterile glove. Scrub the surgical area three more times with disinfectant.

Then, using an autoclave-sterilized scalpel, make an approximately one-centimeter longitudinal skin incision along the interior side of the right femur and use micro scissors and forceps to bluntly separate the biceps femoris and vastus lateralis muscles. Cut the tendon insertion at the femur trochanter to allow free access to the anterolateral part of the bone taking care that the sciatic nerve is preserved, and position the external fixator parallel to the femur. Using a 0.45-millimeter drill bit, manually drill bore holes through cortex.

And starting with the most proximal and distal ends, place ceramic mounting pins into the holes. When all the pins have been placed, humidify the bone with a small volume of sterile saline and use a 0.4-millimeter Gigli wire saw to create a 0.4-millimeter osteotomy through the hole bone between the two inner pins. Flush the osteotomy gap carefully with two milliliters of sterile saline to remove any bone chips from between the two fractured cortices and use a continuous resorbable suture to adapt the muscles followed by adaptation of the skin with interrupted non-resorbable sutures.

Then, clean the surgical area with disinfectant and return the mouse to its cage with monitoring until full recovery. As early as three days after the surgery, insert the external fixator carefully into a custom-made mounting device and place the anesthetized mouse onto a temperature-controlled cradle for introduction into the MRI device. Rigidly attach the external fixator to the mounting device on the four-element head coil, and use a dedicated high-field small animal MRI system operating at 11.7 tesla to apply a proton-density fat-suppressed multi-sliced turbo-spin echo sequence to the fracture.

When all of the images have been acquired, open the data in the appropriate image analysis software and enter the voxel size as 0.05 by 0.05 by 0.35 millimeters cubed. To segment the different tissues in the fracture callus with semi-automatic thresholding based on their intensity, first click on the edit new label field and click Add material. Rename the material to callus, and use the lasso tool to distinguish the callus area from the surrounding tissues based on the hypointense signal from the periosteum.

Click Add to material, then click Add material and rename the material to cartilage. Using the threshold tool, segment the cartilage and Select only current material from the callus. Then, click cartilage and Add to material.

When all of the tissues have been segmented, click Generate surface, apply None for smoothing type and click Surface View to generate 3D reconstructions of the fractured femurs based on the tissue segmentation data. A successful surgical procedure can be confirmed by MRI scan of the fracture showing all four pins in the middle of the femoral shaft with an osteotomy gap between 0.3 and 0.5 millimeters. On day 10 postsurgery, evaluation of the longitudinal scans during the fracture healing process reveals cartilaginous tissue within the middle of the fracture callus and bony tissue at the periphery of the fracture.

Both the cartilaginous and bony tissues increase until day 14. The cartilaginous tissue then decreases until day 21 at which point bony bridging can be observed. After segmentation of the different tissues in the fracture callus as just demonstrated, a whole 3D femur scan can be generated with the mature cortex appearing in gray, the ceramic pins marked in yellow, the callus soft tissue in green, the cartilage tissue in red and the callus bony tissue indicated in purple.

After watching this video, you should have a good understanding of how to apply an external fixator to the mouse femur to create the femur osteotomy and to insert a mouse into an MRI device to ensure a standardized scanning position. Once mastered, the external fixator application and the osteotomy technique can be completed within 20 to 30 minutes if performed properly.