A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

The overall goal of this surgical procedure is to create a drill-hole growth plate injury for the study of repair tissue formation in the evaluation of growth plate repair strategies in a small animal model. This method can help answer key questions related to growth plate injuries, specifically why the bony repair tissue forms after injury and also allows us to test novel treatments that could potentially regenerate the growth plate cartilage. The main advantages of this technique are the relative ease of implementation and the reproducibility of creating bony repair tissue that mimics clinical injuries.

The implications of this technique extend towards the treatment of growth plate injuries as successful outcomes using this model can be translated to similar injuries in the clinic. Generally, individuals new to this technique will struggle with the difficulty of measuring and estimating appropriate drill depth necessary to disrupt the growth plate but without disrupting the articular surface. Before beginning the procedure, use an electric shaver to remove the hair from the entire hind leg of an anesthetized rat from the medial malleolus to the pelvis.

Using calipers, measure and record the tibial length from the anterior tibial plateau to the inferior side of the medial malleolus and use alcohol swabs and povidone iodine soaked gauze to disinfect the entire leg, the abdomen, and the genitalia. Administration of IACUC approved analgesics can be done at this point. Wearing sterile surgical gloves, place a fenestrated sterile surgical drape over the animal, leaving the leg exposed through the central fenestration.

Holding the leg firmly with one hand while pulling the skin tight against the bone, use a scalpel in the other hand to make a one centimeter incision through the skin from the distal end of the medial femoral condyle along the medial anterior aspect of the proximal tibia. Take note of the important anatomical markers, including the growth plate, the growth plate angle, the knee capsule, and the semitendinosus insertion. Then use a scalpel to make 0.5 centimeter incision through the fascia and soft tissues on the medial anterior aspect of the proximal tibia from the growth plate to the bottom of the skin incision and gently scrape away the fascia and soft tissues from the bone.

When all of the tissue has been removed, grasp the leg firmly with one hand and hold a drill equipped with a Steinmann pin perpendicular to the tibial diaphysis. Keeping a firm hold on the leg with the other hand, slowly drill through the tibial cortical bone at the diaphysis to create a two millimeter cortical window that aligns with the distal semitendinosus insertion without drilling through to the other side of the bone. Then dab the finished cortical window with gauze as light bleeding is expected.

To create the growth plate injury, align the end of a 1.8 millimeter dental burr with the proximal tibia where the semitendinosus crosses the knee capsule. To determine the appropriate depth for the burr to fully disrupt the growth plate without disrupting the articular surface, place the end of the dental burr at the knee capsule and follow the burr shaft along the semitendinosus insertion taking note of where the burr aligns with the cortical window. To attain the appropriate drill angle, hold the rotary tool at a less than 30 degree angle with respect to the tibial diaphysis and visualize a line along the dental burr to the growth plate angle.

Now turn on the rotary tool at 10, 000 RPM and enter the cortical window, pushing the tool until until the burr marker aligns with the cortical window and removing the burr once the proper depth has been attained. Dab the cortical window with gauze for about 30 seconds and confirm the appropriate depth of the injury by reinserting the burr into the drill track with the rotary tool off and aligning the burr with the cortical window. Bleeding will interfere with visualization of the dental burr while making the injury so it is important to remeasure the burr to ensure that appropriate depth has been attained.

If the depth is inadequate, push the burr to the desired depth with the tool on. Then use a 10 milliliter syringe equipped with a 23 gauge needle to rinse the drill track with three milliliters of sterile saline and dry the wound with gauze. Inject a biomaterial into the injury site as experimentally appropriate.

Then use 3-0 polyglycolic acid sutures to close the wound and close the skin incision with wound clips. Bony repair tissue has been reported to begin at approximately seven days post-injury, becoming fully developed by 28 days post-injury as visualized by microcomputed tomography. Alcian blue hematoxylin with Orange G-eosin counterstain can also be used to histologically evaluate a variety of repair tissues at different stages of bony bar formation allowing the visualization of different types of repair tissue including mesenchymal, cartilaginous, bony trabeculae, and bone marrow.

Further, the injection of a chitosan microgel into the growth plate injury site can be clearly observed at the injury site allowing subsequent analysis of the effects of the biomaterial on repair tissue composition, limb length, or growth plate measurements. Disruption of the articular cartilage surface creates a larger injury that can introduce articular cartilage into the growth plate injury site complicating the healing process. Disrupting the growth plate at an inappropriate angle or direction results in a non-central injury that results in bony bar formation that is lateral or medial to the desired location.

Once mastered this technique can be completed on both limbs in about 25 minutes. While attempting this procedure it's important to have a surgical assistant to help maintain aseptic surgical technique. After watching this video you should have a good understanding of how to create a drill-hole growth plate injury in a rat model.