Generating a Rabbit Calvarial Defect Model: A Surgical Procedure to Create Round Defects in the Top Portion of the Rabbit Skull to Assess Bone Substitute Materials for Bone Regeneration Capacities

Overview

In this video, we describe the surgical procedure to generate a rabbit calvarial model to evaluate the bone regeneration capacities of bone sample substitution materials of interest.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Surgery

1. Site preparation
   1. Place the rabbit on a heated pad (39 °C) covered by a mattress pad (to avoid burns) on the surgery table. Shave the scalp.
   2. Apply a lubricating gel on the eyes to avoid irritation and dryness. Disinfect the site by scrubbing the skin with povidone iodine (10%). Then drape the rabbit with a sterile surgical drape and cut out an access area for the skull.
   3. Disinfect the surgical site with povidone iodine (10%) for a second time. Apply a lubricating gel on the eyes to avoid irritation and dryness.
   4. Prepare a draped table (sterile drape) on which to place the complete surgical tray.
2. Surgical site opening
   1. Anesthetize locally with a subcutaneous (SC) injection of lidocaine 2% (1 mL) on the skull.
   2. Incise through the skin (with a scalpel) along the calvarial sagittal line, from the orbits to the external occipital protuberance (~4 cm in length). Ensure that the periosteum is incised.
   3. Gently elevate the periosteum (with a periosteal elevator) on both side of the incision. Rinse the site with sterile saline.
3. Cylinder placement
   1. Locate the median and coronal sutures on the skull (Figure 2A, B). 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 (Figure 2C).
   2. Place the first cylinder on the left upper quadrant (left frontal bone), and try to lay the device flat. Fix in the position with strong hand pressure and screw a micro-screw, until resistance is felt. Ensure that the screw head is flush with the surface of the cylinder tab.
   3. Repeat the same procedure on the other tab to fix the cylinder tightly onto the skull. Ensure that the cylinder is hermetically fixed to the bone.
   4. Repeat the procedure on the right upper quarter (right frontal bone), left lower quarter (left parietal bone) and right lower quarter (right parietal bone).
4. Bone drilling of 5 intramedullary holes within the area circumscribed by the cylinders (Figure 1)
   1. Drill an intramedullary hole under saline irrigation (0.8 mm in diameter, ~1 mm in depth) with a round bur on the bone, in the center of the area circumscribed by the cylinder. Ensure that bleeding appears.
   2. Drill two more intramedullary holes along the axis passing through the two tab screws, at the inner edges of the cylinder. Along the perpendicular axe, drill two more intramedullary holes at the inner edges of the cylinder. Ensure that bleeding appears.
   3. Repeat the operation within the three other cylinders.
5. Filling cylinders with material samples and capping (Figure 3)
   1. Prepare the desired bone substitute material according to manufacturer instructions or material specifications.
   2. Fill the first cylinder to the brim with the material sample and close the cylinder by fitting the cap. Repeat the process in the 3 other cylinders.
6. Surgical site closure
   1. Close the skin above the cylinders with an intermittent non-resorbable suture.
   2. Apply a sprayable dressing onto the wound.

2. Post-surgical treatment

1. Stop analgesia and anesthesia (propofol and fentanyl perfusion arrest) supply and check the recovery of autonomous breathing.
2. Stop the ventilation once the animal has recovered autonomous breathing. Maintain the animal under pure oxygen before complete awakening.
3. Inject buprenorphine hydrochloride SC (0.02 mg/kg, 0.03 mg/mL, 0.67 mL/kg) and repeat the injection every 6 h for 3 days as post-surgical analgesia.
4. Transfer the animal into its usual housing with water and complete feeding.

Representative Results

Figure 1: Specifications of PEEK cylinders. Two holes (0.8 mm in diameter) were drilled on the lateral stabilizing tabs for screwing. The positions of the 5 intramedullary holes (0.8 mm in diameter) to be drilled on the skull within the area delimited by the cylinder are marked using red circles.

Figure 2: Representative image of the rabbit skull and placement of the cylinders. Pictures showing the median and coronal sutures on the rabbit skull delineating the left-right parietal and frontal bones (A,B). Placement of the cylinders on both side of the sutures (C). Scale bars = 5 mm.

Figure 3: Representative image of cylinders fixed, filled and caped. Picture showing four cylinders fixed onto the skull of a rabbit with titanium screws. Within the area delimited by each cylinder, 5 intramedullary holes (0.8 mm in diameter, ~1 mm in depth) were drilled under irrigation with a round bur to allow bone cell migration. Cylinders were filled with different bone substitute samples (calibrated volumes) before capping (one closed cylinder is shown only). Scale bar = 5 mm.

Materials

Name	Company	Catalog Number	Comments
Drugs
Fentanyl	Bischel		For analgesia
Ketalar 50mg/ml	Pfizer		Ketamine for anesthesia
Lidohex	Bichsel		Lubricating gel for the eyes
Opsite	Smith and Nephew	66004978	Sprayable dressing
Povidone iodine 10%, Betadine	Mundipharma		anti-infective agent
Propofol 2%	Braun	3538710	For anesthesia
Rapidocain 2%	sintetica		Local anesthesia
Rompun 2%	Bayer		Xylazin for anesthesia
Sevoflurane 5%	Abbvie		For anesthesia
Equipment
Fresenius Vial pilot C	Imexmed		Infusion pump
Heated pad	Harvard Apparatus
Suction dominant 50	Medela
Suction tubing Optimus	Promedical	80342.2
Surgical motor	Schick dental	Qube	Drilling of intramedullary holes
Ventilation	Maquet Servo1
Material
Cylinders and caps	Boutyplast	Customized	composition: PEEK (poly ether ether ketone)
Manual self-retaining shaft	GlobalD	ACT1K
Mobile handle for self-retaining shaft	GlobalD	MTM
Self- drilling screws	GlobalD	VA1.2KL4	cross-drive screws composed by Titanium grade5, ISO 5832-3
Surgical tray
Endotracheal tube Shiley diameter 2,5mm	Covidien	86233	For intubation
Endotracheal tube Shiley diameter 4,9mm	Covidien	107-35G	For intubation
Ethicon prolene 4-0	Ehticon	8581H	Non-resorbable suture
Forceps	Marcel Blanc	BD027R	145 mm
Intubation catheter	Cook medical		Guide for intubation
Round surgical burs	Patterson	78000	0.8 mm in diameter, Drilling of intramedullary holes
Scalpel	Swann-Morton		n°10 and n°15
Scissors	Marcel Blanc	657	180 mm
Syringes Omnifix	Braun	4616057V	5ml, 10ml and 50ml
Venflon G22	Braun	42690985-01	Vasofix safety for the ear iv line