Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

The overall goal of this procedure is to create an externally fixed tibia that can be used for analysis of stabilized fracture healing, distraction, osteogenesis, and healing of a segmental gap defect. First, the rings of the fixator are positioned and held in place by inserting sterile insect pins into the proximal and distal portion of the tibia, and then securing the rings to the sterile insect pins. Next, a fracture or osteotomy is created in the mid diaphysis of the tibia In the final step of the procedure, the third threaded rod is inserted to fully stabilize the proximal and distal rings.

Ultimately, results can be obtained that show mechanisms of fracture healing, distraction, osteogenesis, and bone engraftment through x-ray histologic and molecular analyses. Demonstrating the procedure will be senior researchers, Dr.Diane, who and Y Yuu and postdoctoral fellow Dr.Chelsea Baney from the University of California San Francisco, San Francisco General Hospital. Orthopedic Trauma Institutes laboratory for skeletal regeneration Begin by assembling the custom designed external fixation device.

The fixation frames are made of two aluminum rings stabilized by three stainless steel threaded rods, eight hexagonal nuts and 17 matching bolts. Two aluminum rings are needed per fracture to make the proximal ring loosely screw four hexagonal nuts and matching bolts into the appropriate holes. To make the distal ring screw in four nuts and bolts again without tightening fully, then place two of the long threaded rods that will stabilize the fixator to the distal ring in the holes located at 12 and six o'clock, and attach with two hexagonal nuts.

Anesthetize a mouse of the desired age in sex by an intraperitoneal injection of ketamine and meine. And confirm complete anesthesia using a toe pinch test. After removing hair and washing with antiseptic, apply eye lubricant, then place the mouse on a heating pad for the remainder of the procedure.

Place the proximal ring onto the leg with the nuts facing distally and move the ring above the knee joint. Transfix the proximal and distal metaphysis of the left tibia using four 0.25 millimeter sterile insect pins by attaching the pins to a drill and drilling them through the bone. Orient the pins perpendicular to the long axis of the tibia and at 90 degrees to each other, leaving at least 10 millimeters between the proximal and distal pins.

Secure the pins to the proximal ring, keeping the tibia positioned centrally within the aluminum ring. Position the second ring below the distal pins. Secure the two rods to the proximal ring and then secure the pins to the ring using the hexagonal nuts.

As before, create the transverse fracture by three point bending with an appropriate device. Rest the postal lateral surface of the tibia across the blocks. Raise the weight to a predetermined height along the drop arm and drop the weight so that it contacts the anterolateral aspect of the tibia.

To produce the blunt fracture, confirm the fracture by x-ray. Next, attach the last stainless steel threaded rod to the rings starting from the distal end. Insert the rod and thread a nut onto the distal ring to secure the rod.

Then put another nut onto the rod so that the proximal ring is positioned on top. Ensure that the two rings will be held at equal distances and are not straining the pins. Secure the proximal ring to the rods with a third nut administer.

Add aamaz all to reverse anesthesia and buprenorphine for analgesia and monitor the animals until they are responsive and ambulatory. Then administer subcutaneous injections of buprenorphine for analgesia and again, four hours later for standard distraction osteogenesis. Using this model, place the fixators onto the tibia, create fracture and leave them alone for a latency period of five days.

After five days, perform the distraction osteogenesis by turning the nuts on the stabilizing rods. One quarter turn every day for seven days. The animals are monitored closely postoperatively for the need for additional analgesia.

Bony bridging is observed by 10 days, and complete consolidation is observed by 27 days after the completion of distraction. After administering anesthesia, removing hair with a depilatory and cleaning the skin with antiseptic, apply the fixators as previously described. Place the mouse under the dissecting microscope to visualize the medial tibia and then make a five to seven millimeter incision with a number 10 blade on a scalpel.

Gently transect and separate the muscle to expose the mid diaphysis of the tibia. Using the point of a pair of scissors slowly snip away a three millimeter segment of tibia being careful not to displace the bone with a large mechanical force. Then remove any residual bone fragments with forceps.

After examining the surgical site for bone fragments, replace the muscle around the blunted ends of the tibia. Close the incision on the skin with two to three sutures of 6.0 poly monofilament, and then insert the third rod to stabilize the external fixator. As shown earlier, reverse anesthesia administer analgesics and continue to monitor the animals for pain.

As previously described when properly applied, the external fixators provide highly rigid stability of the closed tibial fracture with excellent reduction as shown in this radiograph taken after fracture with the well-aligned bone segments indicated by the arrowhead fractures stabilized using this method heal primarily via intramembranous ossification shown here using tri chrome staining, the stabilized fracture shows some new bone at the fracture site. In contrast, if the fracture is not stabilized, a large cartilage callus is formed in the fracture gap, and this is replaced by bone through the process of endochondral ossification After its development. This technique paved the way for researchers in the field of orthopedics to explore mechanisms of stable fracture healing in mice.