Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

This femur osteotomy model can help researchers study the process of fracture healing and evaluate how a drug could influence fracture healing. It is a simple and easy to follow rodent model that can help researchers identify if a drug has bone-healing properties and understand its mechanism of action. Even though it mainly focuses on observing and analyzing fracture healing, it can also give insights into disease pathogenesis.

This method can guide future research on bone-healing in health and disease. It can be used to evaluate the effect of potential osteoanabolic drugs. Inducing a proper fracture line and fracture fixation with with a K-wire are challenging.

A couple of practice sessions should help. The simplicity and reproducibility of this method are evident in its visual demonstration Pawan Pagaku, a junior resident from my laboratory, will help to demonstrate the procedure. To begin place the wax block containing wax up to a depth of 2.5 centimeters on the operating table and cover it with sterile drapes.

After making a one centimeter vertical skin incision on the lateral side of the right thigh, expose the vastus lateralis muscle by separating the deep fascia using Metzenbaum scissors. Split the vastus lateralis in line with the muscle fibers until the shaft of the femur is reached. Then, use the periosteal elevator to free the bone from the muscles attached to it.

After injecting local anesthesia in and around the periosteum, create an indentation in the middle third of the shaft of the femur using the number 15 surgical blade. Place a chisel on the indentation and gently tap the chisel with a hammer to completely fracture the bone in the middle third of the shaft. Internally fix the fracture using a one-millimeter sterile K-wire held with the help of a battery operated power drill.

Pass the K-wire into the medullary canal of the distal fragment through the fracture site. Then, drill out the K-wire through the distal end of the femur. After reducing the fracture, advance the K-wire from the distal end into the canal of the proximal fragment until it obtains purchase in the trochanteric region.

Cut off the distal part of the K-wire protruding through the skin using a wire cutter. Bend the tip of the K-wire to around 90 degrees using pliers and use a gauze bandage soaked in Betadine for pin site dressing. The K-wire acts as an intramedullary splint for keeping the fracture in a reduced position.

Apply pressure on the bleeding area using sterile gauze or artery forceps to stop any bleeding. Ensure complete hemostasis before closing the skin using a 4-0 nylon suture. Clean the wound with Betadine and cover it with sterile gauze and micropore adhesive tape.

For postoperative care, return the rats to their cages and continue giving a standard semi-synthetic diet as well as antibiotics and analgesics intraperitoneally for five days after the procedure. Finally, assess bone healing by X-ray of the fractured site once weekly. X-rays of the rat's femur with the K-wire NC2 before inducing the fracture, and one day after surgery are shown here.

The fracture healing is monitored radiologically by taking sequential weekly X-rays of the operated site to assess callus formation. The fracture remains reduced and immobilized with the intramedullary K wire. After sacrifice at a predetermined time point, the femur was carefully preserved in formalin, followed by bone decalcification.

Bone with callus was obtained after optimal decalcification. The intact callus, and sagittal section of the callus are shown here. The callus is intermittently assessed to ensure optimal decalcification before evaluating it using other techniques.

Low magnification and high magnification images of the hematoxylin and eosin stained section of the fracture site are shown here. The stained sections of the fracture site show a hard callus with the formation of cartilage and new bone. The fractured end of the bone and the second cortical region can be seen here.

The periosteum most pain-sensitive part of the wound, and it is essential to infiltrate the periosteum at the fracture site with a local anesthetic before the osteotomy. It can be followed by immunohistochemical and radiological analysis of the callous, micro-CT for quantitative histomorphometry, analyzing the biomechanical strength of the callus analyze of serum. This technique is useful to determine the osteogenic potential, mechanism of action, and comparative analysis of promising osteoanabolic drugs.