A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

This surgical protocol uses a consistent and reproducible externally fixed segmental defect model to evaluate the healing mechanisms of bone and the use of potential regenerative therapies. We have streamlined a challenging surgery for the creation and stabilization of a segmental bone defect model that is accessible and cost effective while remaining appropriate for longitudinal healing studies. This technique is particularly translatable to the treatment of traumatic critical-sized diaphysial bone defects that may benefit from stabilization with external fixation, although the model requires further study.

To prevent a fracture while drilling the pins, use high rotations per minute and a low pressure to puncture the lateral cortex, then decrease the rotations per minute to advance. After preparing the scaffold from a bone morphogenetic protein tube bone graft kit, according to the manufacturer’s instructions, use sterile scissors and a sterile ruler to trim the collagen sponge to fit a five by three by three millimeter defect. Then use a syringe to distribute the bone morphogenetic protein-2 solution evenly over the surface of the sponge, until the entire volume of solution is absorbed.

Next, shave the area around the hind leg of an anesthetized rat using the 13th rib, hind paw, and dorsal and ventral midlines as margins, followed by four sequential alternating scrubs of the exposed skin with sterile gauze soaked in 10%povidone-iodine and 70%ethanol. To induce the femoral defect, extend the shaved leg through a fenestrated clear sticky drape, and cover the surgical bench in sterile towels to create a sterile field. Palpate the femur and use a number 15 blade to create an anterolateral incision through the skin extending from the patella to the greater trochanter at the proximal femur.

Carefully incise the lateral leg fascia along the intermuscular septum to separate the vastus lateralis muscle of the quadriceps anteriorly from the hamstrings posteriorly until the lateral femur is exposed. Keeping a number 15 scalpel blade parallel against the contour of the bone surface, gently cut the muscle away from the underlying bone to perform a careful, atraumatic circumferential soft tissue dissection, and start on the lateral surface, expose the femur at its mid-diaphysis. Use a periosteal elevator to lift the muscle away from the exposed bone as it is dissected, and proceed around the femoral shaft until seven to 10 milliliters of central diaphysis has been cleared of soft tissue on all sides.

Next, place one pin just at the level of the lateral epicondyl, followed by placement of a jig flush to the lateral distal femur, and insert one one-millimeter threaded tip Kirschner wire. Next, maintaining the position of the jig on the bone, identify where the most proximal pin will enter the bone based on the jig holes. Carefully incise parallel to the fibers of the gluteal tendon as needed to create a small gap in the tissue for the proximal pin to pass through, minimizing iatrogenic damage to the tendon.

Drill a one-millimeter non-threaded k-wire into the gap, and compare the height of the drilled pin with the loose pin to confirm that the pin engages both cortices. Maintain the position of the jig in contact with the bone and drill two one-millimeter threaded k-wires, one on either side of the future defect site, ensuring that the pins engage both cortices, and place the external fixator bar level one centimeter above the skin. Screw tightly, locking the bar in place, and clip the excess pin lengths.

Place a small curved retractor around the anterior and posterior femur to protect the surrounding soft tissue, muscle, and neurovascular bundle, and use an approximately five millimeter sagittal oscillating saw blade, and light, even pressure, to very carefully create a five millimeter segmental defect through the mid-diaphysis. Flush the wound with 10 milliliters of 0.9%normal saline after creating the defect, and administer 0.1 milliliters of 0.25%bupivacaine with epinephrine to the wound as an analgesic and vasoconstrictor. Insert the bone morphogenetic protein-2 soaked sponge scaffold into the defect, and use a 4-0 absorbable suture in the simple interrupted pattern to close the muscle plane.

Then close the skin layer using a 4-0 absorbable suture in a running subcuticular pattern, then skin glue to close any gaps around the protruding pins, and obtain an anterior-posterior radiographic image of the femur immediately, and four and 12 weeks after surgery. Negative control defects containing only a collagen sponge show no evidence of bridging osteogenesis between the proximal and distal bone edges. A small amount of new bone remodeling can be seen directly adjacent to the cut femur edge, while the defect itself shows a lack of bony material, the presence of cartilage, and some residual hematoma.

Defects containing bone morphogenetic protein-2 soaked sponge demonstrate significant bone healing as early as four weeks after surgery, as evidenced by the radiopaque callous bridging across the defect. By 12 weeks, significant new mineral deposition has formed throughout the defect. Significant new periosteal bone can be observed in the callous extending from the cut femur edge and the spicules of woven and lamellar bone have developed throughout the defect.

Further, cartilage deposition is not seen. Imaging via In Vivo Imaging System after the removal of the external fixator allows visualization of the bioluminescent transfected cells within the defect. For example, cells in the medullary cavity luminesce after transfection with complexed mRNA encoding for Gaussia luciferase, and could be used to measure biological changes in the expression of a gene or protein of interest during the healing process.

Pin placement is important for ensuring a stable, lasting external fixation. The pins should be directly perpendicular to the femur, and parallel to each other, to minimize breakage and infection. Using this model, biological mechanisms, such as specific protein expressions, may be monitored to better understand healing, and different scaffold biomaterials or therapies may also be tested for therapeutic efficacy.

We hope this technique will allow future researchers to more efficiently and effectively study orthopedic healing with external fixation so that their findings may be translated to improved clinical successes. Avoid inhaling anesthetic gases, wear proper personal protective equipment when handling formalin and EDTA, and take care when handling surgical instruments, particularly the drill and oscillating bone saw.