Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

Here we present a surgical protocol for rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. The basic principle of this rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth support and neovascularization support.

The model is highly significant to assess bone regeneration as it measures the ectopic growth of new bone tissue above mature bone. When compared to traditional models, in which new bone regenerates to fill defect that was artificially created to finally go back to its initial level. By using big cylinders fixed on rabbit skulls, osteoconduction, osteoinduction, osteogenesis, and vasculargenesis induced by the materials may be evaluated either on live or sacrificed animals.

Demonstrating the procedure will be Laurin Marger, a research associate from our laboratory, and myself. After sedating the animals, place an intravenous canula into the marginal vein from the ear and keep it closed until intubation is complete. Place the rabbit in the prone position and maintain its head in vertical extension.

Anesthetize the trachea locally by spraying 10%lidocaine. Slide a small diameter endotracheal tube into the rabbit's trachea until airflow can be heard in the tube. This will open the larynx and facilitate the insertion of the definitive tube.

Next, insert a guide into the tube to fix the position of the tube into the trachea. Remove the small diameter tube and slide the definitive endotracheal tube on the guide. Remove the guide and inflate the balloon at the end of the endotracheal tube to seal and block the device into the trachea.

Continuously profuse fentanyl to induce analgesia. Two to four milligrams per kilogram of propofol to induce anesthesia and four milliliter per kilogram per hour of ringers acetate to maintain isovolumetric conditions. Immediately ventilate the animal with 3%sevoflurane in pure oxygen.

First, place the rabbit on a heated pad set to 39 degrees Celsius on the surgery table, and place the rectal temperature probe. Shave the scalp. Scrub the skin with 10%providone iodine to disinfect the site.

Then, drape the rabbit with a sterile surgical drape and cut out an access area of the skull. Disinfect the surgical site with 10%providone iodine a second time. To begin, anesthetize locally with a subcutaneous injection of lidocaine on the skull.

Use the scalpel to incise through the skin along the calvarial sagittal line from the orbits to the external occipital protuberance, making sure that the periosteum is incised. Use a periosteal elevator to gently elevate the periosteum on both sides of the incision. Rinse the site with sterile saline.

First, locate the media and coronal sutures on the skull. Note that these anatomical lines form a cross. The cylinders will be placed in each of the quadrants defined by the cross, ensuring that the edge of the cylinder is not over the suture.

Place the first cylinder on the left upper quadrant laying the device as flat as possible. Fix the cylinder's position with strong hand pressure and screw in a micro screw until resistance is felt. Ensure that the screw head is flush with the cylinder tab.

Fix the cylinder's other tab the same way to secure the cylinder tightly on to the skull. Ensure that the cylinder is hermetically fixed to the bone. Then repeat this entire procedure to secure a cylinder onto each of the other three quadrants.

To begin, drill an intermedullary hole under saline irrigation with a round burr on the bone on the center of the area circumscribed by the cylinder. Ensure that bleeding appears. Next, drill two more intermedullary holes along the axis passing through the two tab screws at the inner edges of the cylinder.

Along the perpendicular ax, drill two more intermedullary holes at the inner edges of the cylinder. Repeat this drilling process for the three other cylinders. Then, fill the first cylinder to the brim with the material sample.

Close the cylinder by fitting the cap. Repeat this filling process for the other three cylinders. Close the skin above the cylinders with an intermittent non-resorbable suture and apply a sprayable dressing onto the wound.

After this, stop the analgesia and anesthesia supply and check for the recovery of autonomous breathing. Once the animal has recovered autonomous breathing, stop the ventilation. Maintain the animal under pure oxygen before complete awakening.

Inject buprenophine hydrochloride subcutaneously. Repeat the injection every six hours for three days as post surgical analgesia. Allow the animal to awake completely before transferring it to its usual housing with water and complete feeding.

The model described in this video is dedicated to the assessment of osteoconduction, osteoinduction, osteogenesis, and neovascularization in bone substitutes. Once the surgery is completed, bone growth may be monitored at different time points by using bone tomography on live animals. Additional analysis requires the animals to be sacrificed.

After euthanasia, samples are sectioned and the cylinders are carefully removed. Biopsies are then fixed with a solution of 4%formaldehyde in PBS. After this, microtomography may be use to assess bone growth.

Samples may also be processed for histological staining. Histomorphometric analysis and specific stainings are then possible to complete the analysis more specifically. As this protocol is a surgical method, all the steps are critical and must be followed properly.

It is important to be trained for animal experiments, especially in rabbit handling and anesthesia. Apart from the methods described, cells may be analyzed more specifically by using flow cytometry. Material evolution, bone tissue maturation, and mechanics may also be evaluated by using nano indentation, for example.

With the aid of this model, many assessments were performed. As for example, for titanium or ceramic cages, GBM membranes, osteogenic factors, newborn substituents or the mechanism of neovascularization during the bone regeneration process.