An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

The overall goal of this protocol is to reproduibly and efficiently induce femoral distraction osteogenesis in a rat model to assess functional bone regeneration. This method can help answer key questions in the osteogenesis medicine field about determining and shortening the consolidation period or about identifying the mechanism of bone repair. The main advantage of this technique is vital to who made external fixator and to guide who is doing the surgery, provide a stable environment for optimal bone regeneration.

The implications of this technique extend to all therapeutic upper and lower limb lengthening or of bone defect that occur after bone tumor excision surgery. The demonstration of the procedure is done by Edouard Lamy, a pharmacy lecturer from my laboratory. Begin by placing an anesthetized 12-week old male Sprague Dawley rat in the left lateral position and removing the hair from the right hind limb.

After applying eye ointment, disinfect the surgical area with Povidone-iodine solution and place the animal in the supine position. With the external fixator positioned along the mediolateral axis, use a marker to make one point at the knee and a second point at the hip along the medial line of the sagittal plane. Connect the two points and use a scalpel to make a skin incision along the line, cutting between the biceps femoris and the vastus lateralis until the femur is fully exposed.

Lift the periosteum and use a rougine to disconnect the soft tissue from the bone. Insert pins into the most proximal and distal holes of the external fixator. If both pins can be anchored, use two Senn's retractors to open the muscles and tighten the clamp of a drilling guide in the middle of the femur, starting with the proximal and distal holes, and finishing with the middle two holes, pass a metal 0.6 millimeter drill bit through the bone to create four guide holes.

Using a 0.8-millimeter half-threaded pin, work the pin back and forth to enlarge the four pre-holes, taking care to stay perpendicular to the femur. To implant the pins, use a needle holder to grasp the head of each 1-millimeter half-thread pin and individually sink the pins to enlarge the guide holes. Use a Senn's retractor to confirm that the pins penetrate both of the cortices without protruding more than 1 millimeter.

When the pins are in place, connect the external fixator to the half-threaded pins, making sure that the offset is about 6 millimeters to allow easy stitching and good rigidity. Then secure the four locking screws so that the external fixator is locked to the pins. Using a Piezotome, perform an osteotomy between the two central pins.

Then use a Mayo-Hegar Needle Holder, a resorbable 5-0 suture thread and a continuous stitch to close the wound. Immediately after the surgery, x-ray the animal to check the depth of the pins and the alignment of the osteotomized extremities along the long axis. Then allow rat to recover fully with monitoring.

After one week, x-ray the limb to recheck the positioning of the pins and the alignment of the bone segments. To perform the distraction, manually turn the square nut one-half turn clockwise every 12 hours for 10 days. X-ray the limb halfway through and at the end of the distraction process to monitor the positioning of the pins and the alignment of the bone segments.

At the end of the distraction, x-ray the limb weekly, removing the external fixator on day 64. On day 66, use a scalpel to make a skin incision through the surgery scar from the top of the hip to the front of the knee. Cut between the biceps femoris and the vastus lateralis until the femur is fully exposed, disconnecting the muscle from the bone as much as possible.

Cut all of the ligaments of the knee, dismantle the articulation, and cut the joint capsule of the hip. Clean the bone thoroughly without removing the pins, using a scalpel to remove all of the soft tissue. When the contralateral femur has been cleaned, x-ray both femurs.

Remove the pins and store the femurs at minus 20-degree Celsius for micro-computed tomography scan analysis. In this representative experiment, x-ray imaging from immediately after the surgery through the end of the consolidation, revealed that the half-threaded pins within the femur pins remained parallel, well preserved, and unloosened throughout the procedure, indicative of a stable anchorage. Moreover, the osteotomized extremities had clearly been well aligned along the longitudinal axis of the bone during the distraction osteogenesis process.

At the end of the latency period, no calcified areas were visible near the osteotomized extremities. At the end of the distraction period, a few calcified areas were visible close to the pre-existing cortices. After 28 days of consolidation, the non-calcified region of the gap between the osteotomized extremities was smaller, and a periosteal callus could be observed near the gap level with the pins.

After 47 days of consolidation, the regenerating callus was completely bridged. After removal of the external fixator and two days of physiological weightbearing, the animals displayed a physiological gait and no evidence of fracture could be observed. 3D micro-computed tomography analysis of the serial longitudinal sections of the regenerating callus demonstrated that calcified bridging was always present with a continuous outer cortical observed at the periphery of the regenerating callus.

After 49 days of consolidation, no fracture was observed despite the persistence of a less mineralized region at the center of the regenerated callus. Once mastered, this surgical technique can be completed in 45 minutes if it is performed properly. While attempting this procedure, it is important to remember to maintain a sterile environment during the surgery and to thoroughly monitor the animal after the surgery.

After it's development, this technique pave the way for researcher in the field of medicine to improve the distraction osteogenesis technique, particularly for shortening the consolidation period in the clinical practice. Following this procedure, other methods like adding growth factors or build materials can be performed to answer additional questions such as, can the consolidation of the bone be improved. After watching this video, you should have a good understanding of how to perform femoral lengthening in a rat model using these techniques as a guide to ensure parallelism between the pins.