A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

The overall goal of this surgical technique is to provide standardized conditions for the analysis of fracture healing in mice. This method can help answer key questions in fracture healing research, such as the role of different growth factors in the mechanisms of bone repair. The main advantage of this procedure is that it can be used in a minimally invasive technique and provide standardized biomechanical conditions for the study of endochondral fracture healing in mice.

In the following, Philipp, a student, will demonstrate the surgical procedure. Begin by ensuring that all required sterile surgical instruments and materials are positioned on an operation cloth directly adjacent to the small animal operation table. For this study, 12 to 14-week-old male CD-1 mice were used.

After inducing anesthesia using an approved method, confirm anesthetization by the lack of response to a toe pinch. Apply eye lubricant to protect the eyes from drying during anesthesia, and place the anesthetized mouse under a heat radiator to keep the body temperature constant. Before surgery shave the entire right hind leg and apply a depilatory cream.

After five minutes remove the cream and clean the leg with water. Then apply a disinfecting solution with 96%alcohol. Place the mouse in the supine position on the small animal operating table.

Under aseptic conditions, bend the right knee to allow for an anterior approach to the condyles of the knee. Use a scalpel blade to perform a five millimeter medial parapatellar incision at the right knee. Mobilize the patellar ligament carefully with the scalpel blade and the swab.

Then use fine forceps to shift the patella laterally to expose the intercondylar notch of the femur. Open the intercondylar notch exactly in the middle of the femur between both condyles. Use a hand drill with a 0.5 millimeter centering drill bit, and start drilling at a slow speed and a 45 degree offset ventrally to the femur axis.

During drilling, continuously decrease the angle to zero degrees offset until it is parallel with the bone axis of the femur. Stop drilling when a depth of one millimeter is reached. Insert a 27 gauge needle into the intramedullary cavity over the whole length of the femur and ream the intramedullary cavity of the femur manually using a rotary motion.

Then push the needle forward to perforate the cortical bone at the greater trochanter proximally. Remove the 27 gauge needle and apply the guide wire through the distal part of the femur. Then use a size 15 scalpel blade to make a skin incision proximally over the guide wire.

Keep the guide wire in place while pushing it forward until both ends of the guide wire are outside. To create a defined closed fracture using the guillotine, place the mouse in a lateral position with the right leg under the guillotine. Make sure that the diaphyseal part of the femur is placed in the middle of the guillotine.

Drop the 200 gram weight from the defined distance of 7.6 centimeters. Use the X-ray device to control the fracture configuration and position as well as the position of the guide wire. Use the nose at the distal end of the intramedullary screw to connect it to the 0.2 millimeter guide wire.

Insert it into the femur under continuous pressure and clockwise rotation. Shear off the drive shaft when the sufficient torque is achieved, and then remove the guide wire proximally. Reposition the patella, and fix the patella tendon to the muscles with one single suture using a 5-0 synthetic monofilament nonabsorbable polypropylene suture.

Use single sutures of the same material and size to close the wound. Use the X-ray device to control the reduction of the fragments and the screw position radiologically. Keep the post-surgical animal under the heat radiator until recovered from anesthesia.

Observe the mouse until it has regained sufficient consciousness to maintain ventral recumbency. Return all postoperative animals to single cages in the animal facility. Monitor the animals carefully every day, and provide postoperative analgesia using approved methods for the first three days after surgery.

Radiological analyses after two weeks showed an obvious formation of callus tissue bridging the fracture gap. After five weeks the fracture was healed and the periosteal callus was almost completely remodeled. Histological analyses of the callus and the fracture zone after two weeks showed typical distribution of endochondral healing with carticle tissue built during the chondrogenic process and woven bone.

After five weeks the cartilage tissue disappeared and the woven bone was converted to the lamellar bone to reconstitute the normal anatomical and load-carrying properties of the bone. Biomechanical analyses after two weeks indicated a bending stiffness of 37%compared to the contralateral unfractured bone. After five weeks the bending stiffness was almost 100%indicating complete healing.

Once mastered, this technique can be done in 20 minutes if it's performed properly. After watching this video, you should have a good understanding of how to use this technique as a standardized model for fracture healing research.