Name: calvarial model of bone augmentation in rabbit for assessment of bone growth and neovascularization in bone substitution materials
Uploaded: 2019-08-13T14:30:00
Description: Watch this Scientific Journal Video about Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials at JoVE.com

The authors are indebted to Geistlich AG (Wolhusen, CH) and the Osteology foundation (Lucerne, CH) (grant n&#176;18-049) for their support, as well as Global D (Brignais, FR) for providing the screws. A particular thanks goes to Dr B. Schaefer from Geistlich. We are also grateful to Eliane Dubois and Claire Herrmann for their excellent histological processing and their precious advices. Finally, we warmly acknowledge Xavier Belin, Sylvie Roulet and the entire team of Pr Walid Habre, &#8220;experimental surgery Dpt&#8221; ,for their remarkable technical assistance.

In line with Swiss legal requirements, the protocol was approved by an academic committee and supervised by the cantonal and federal veterinary agencies (authorizations n&#176; GE/165/16 and GE/100/18).
1. Specific devices and animals
<ol>
	<li>Cylinders
	<ol>
		<li>Machine cylinders with lateral stabilizing tabs out of PEEK to have inner diameter of 5 mm, outer diameter of 8 mm and a height of 5 mm (Figure 1).</li>
		<li>Machine PEEK caps with ...

The model described herein is simple and should be developed quite easily as long as all the steps are followed and the equipment is suitable. As the protocol described is a surgical method, all the steps appear critical and must be followed properly. It is critical to be trained for animal experiments, especially in rabbit handling and anesthesia. Do not hesitate to ask for professional anesthetist and veterinary help. It is critical to insist on the daily visual monitoring of animals before and after suture removal. Ev...

The calvarial model of bone augmentation was developed in the 90&#8217;s with the aim to optimize the concept of guided bone regeneration (GBR) in the oral and craniofacial surgical domain. The basic principle of this model is to grow new bone tissue vertically on top of the cortical part of the skull. To do so, a reactor (e.g., titanium -dome, -cylinder or -cage) is fixed onto the skull to protect the bone regeneration conducted by a graft (e.g., hydrogel, bone substitute, etc.). With the aid of this model, titanium or ceramic cages1,2,3,4,5,6, GBR membranes7,8,9,10, osteogenic factors11,12,13,14,15,16,17, new bone substitutes12,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or the mechanism of neovascularization during the bone regeneration process30 were assessed.
From a translational point of view, the calvarial model represents a one-wall defect that can be compared to a class IV defect in the jaw31. The aim is to grow new bone above a cortical area, without any lateral support from endogenous bone walls. The model is thus extremely stringent and assesses the real potential of vertical osteoconduction over the cortical part of the bone. If the model described herein is primarily dedicated to the assessment of osteoconduction in bone substitutes, osteogenesis and/or osteoinduction may be also assessed, as well as vasculogenesis1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30.
Essentially for ethical, practical and economic reasons, the calvarial model was developed in the rabbit in which the bone metabolism and structure are quite relevant when compared to human32. Of the 30 references cited above, 80% used the rabbit calvarial model1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,17,22,23,26,27,28,29,30,33, thus demonstrating the relevance of this animal model. In 2008, the Busenlechner group transferred the calvarial model to the pig, to allow the comparison of eight bone substitutes simultaneously20 (as compared to two bone substitutes with the rabbit). On the other hand, our group transferred the rabbit calvarial model to sheep. In brief, titanium domes were placed on sheep skulls to characterize the osteoconduction of a new 3D-printed bone substitute. These studies allowed us to develop and master the calvarial model and its analysis16,21.
The last three studies cited16,20,21, together with several other investigations12,17,18,19,22,23,24,26,27,28,29, confirmed the great potential of the calvarial model as a screening and characterization model. However, even though the results obtained were quite satisfactory, they also pointed out some limitations: (1) The use of titanium domes, which prevented X-ray diffusion and in turn live micro-CT use. These could not be removed before histological processing, forcing the researchers to embed the samples in poly(methyl methacrylate) resin (PMMA). The resulting analyses were therefore largely limited to topography. (2) High financial costs especially because of the cost of the animals, and costs related to the logistics, maintenance and the surgery of the animals. (3) Difficulties to obtain ethical approvals for large animals.
A recent study by Polo, et al.26 largely improved the model on the rabbit. Titanium domes were replaced by closable cylinders that could be filled with a constant volume of material. Four of these cylinders were placed on rabbit skulls. At completion, the cylinders could be removed so that biopsies were metal-free, introducing much more flexibility concerning sample processing. The rabbit calvarial model became attractive for simultaneous testing with lower costs, easy animal handling and facilitation of sample processing. Taking advantage of these recent developments, we have further improved the model by replacing titanium with PEEK to produce cylinders, thereby allowing X-ray diffusion and the use of microtomography on live animals.
In this article, we will describe the anesthesia and surgery processes and show examples of outputs that may be obtained using this protocol, i.e., (immuno-) histology, histomorphometry, live and ex vivo microtomography to evaluate the mechanisms of bone regeneration and quantify the new bone synthesis supported by bone substitute materials.

<table>
	<tbody>
		<tr>
			<td>Drugs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxacine Baytril 10%</td>
			<td>Bayer</td>
			<td></td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Bischel</td>
			<td></td>
			<td>For analgesia</td>
		</tr>
		<tr>
			<td>Ketalar 50mg/ml</td>
			<td>Pfizer</td>
			<td></td>
			<td>Ketamine for anesthesia</td>
		</tr>
		<tr>
			<td>Lidohex</td>
			<td>Bichsel</td>
			<td></td>
			<td>Lubricating gel for the eyes</td>
		</tr>
		<tr>
			<td>Opsite</td>
			<td>Smith and Nephew</td>
			<td>66004978</td>
			<td>Sprayable dressing</td>
		</tr>
		<tr>
			<td>Povidone iodine 10%, Betadine</td>
			<td>Mundipharma</td>
			<td></td>
			<td>anti-infective agent</td>
		</tr>
		<tr>
			<td>Propofol 2%</td>
			<td>Braun</td>
			<td>3538710</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Rapidocain 2%</td>
			<td>sintetica</td>
			<td></td>
			<td>Local anesthesia</td>
		</tr>
		<tr>
			<td>Ringer-acetate</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td>Volume compensation</td>
		</tr>
		<tr>
			<td>Rompun 2%</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazin for anesthesia</td>
		</tr>
		<tr>
			<td>Sevoflurane 5%</td>
			<td>Abbvie</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Sintetica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temgesic</td>
			<td>Reckitt Benckiser</td>
			<td></td>
			<td>Buprenorphine hydrochloride, analgesia</td>
		</tr>
		<tr>
			<td>Thiopental Inresa</td>
			<td>Ospediala</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Xylocaine 10% spray</td>
			<td>Astra Zeneca</td>
			<td></td>
			<td>For intubation</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fresenius Vial pilot C</td>
			<td>Imexmed</td>
			<td></td>
			<td>Infusion pump</td>
		</tr>
		<tr>
			<td>Heated pad</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suction dominant 50</td>
			<td>Medela</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suction tubing Optimus</td>
			<td>Promedical</td>
			<td>80342.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical motor</td>
			<td>Schick dental</td>
			<td>Qube</td>
			<td>Drilling of intramedullary holes</td>
		</tr>
		<tr>
			<td>Ventilation</td>
			<td>Maquet Servo1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cylinders and caps</td>
			<td>Boutyplast</td>
			<td>Customized</td>
			<td>composition: PEEK (poly ether ether ketone)</td>
		</tr>
		<tr>
			<td>Manual self-retaining shaft</td>
			<td>GlobalD</td>
			<td>ACT1K</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobile handle for self-retaining shaft</td>
			<td>GlobalD</td>
			<td>MTM</td>
			<td></td>
		</tr>
		<tr>
			<td>Self- drilling screws</td>
			<td>GlobalD</td>
			<td>VA1.2KL4</td>
			<td>cross-drive screws composed by Titanium grade5, ISO 5832-3</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical tray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal tube Shiley diameter 2,5mm</td>
			<td>Covidien</td>
			<td>86233</td>
			<td>For intubation</td>
		</tr>
		<tr>
			<td>Endotracheal tube Shiley diameter 4,9mm</td>
			<td>Covidien</td>
			<td>107-35G</td>
			<td>For intubation</td>
		</tr>
		<tr>
			<td>Ethicon prolene 4-0</td>
			<td>Ehticon</td>
			<td>8581H</td>
			<td>Non-resorbable suture</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Marcel Blanc</td>
			<td>BD027R</td>
			<td>145 mm</td>
		</tr>
		<tr>
			<td>Intubation catheter</td>
			<td>Cook medical</td>
			<td></td>
			<td>Guide for intubation</td>
		</tr>
		<tr>
			<td>Needlle holder</td>
			<td>Marcel Blanc</td>
			<td>BM008R</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles BD Microlance3</td>
			<td>Becton Dickinson</td>
			<td>300300/304622</td>
			<td>26G; 18G</td>
		</tr>
		<tr>
			<td>Periosteal</td>
			<td>HU-Friedy</td>
			<td>P9X</td>
			<td></td>
		</tr>
		<tr>
			<td>Round surgical burs</td>
			<td>Patterson</td>
			<td>78000</td>
			<td>0.8 mm in diameter, Drilling of intramedullary holes</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Swann-Morton</td>
			<td></td>
			<td>n&#176;10 and n&#176;15</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Marcel Blanc</td>
			<td>00657</td>
			<td>180 mm</td>
		</tr>
		<tr>
			<td>Syringes Omnifix</td>
			<td>Braun</td>
			<td>4616057V</td>
			<td>5ml, 10ml and 50ml</td>
		</tr>
		<tr>
			<td>Venflon G22</td>
			<td>Braun</td>
			<td>42690985-01</td>
			<td>Vasofix safety for the ear iv line</td>
		</tr>
	</tbody>
</table>

calvarial model of bone augmentation in rabbit for assessment of bone growth and neovascularization in bone substitution materials

The basic principle of the 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 and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

The model described herein is dedicated to the assessment of osteoconduction in bone substitutes. Osteogenesis and-or osteoinduction of bone substitutes either (pre-)cellularized or loaded with bioactive molecules may be also assessed, as well as vasculogenesis1,2,3,4,5,6,<sup class...

Watch this Scientific Journal Video about Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials at JoVE.com

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

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

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.

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