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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

The calvarial model of bone augmentation was developed in the 90’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.

Protocol

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° GE/165/16 and GE/100/18).

1. Specific devices and animals

  1. Cylinders
    1. 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).
    2. Machine PEEK caps with a design allowing to clip precisely onto the top of the cylinder (thickness 1 mm).
    3. Sterilize PEEK cylinders and caps by autoclaving before surgery.
  2. Screws
    1. Use self-drilling micro screws (made of commercial pure titanium (grade 5)) to fix the cylinders (1.2 mm in diameter, 4 mm in length). Sterilize by autoclaving before surgery.
  3. Animals
    1. Purchase three-month-old New Zealand white rabbits (male or female), weighing ~2.5 kg each.
      NOTE: We obtained rabbits by breeding at the University of Geneva.

2. Surgery

  1. Surgical tray
    1. Keep scalpels, scissors, two forceps, periosteal elevator, syringes (1, 2, 5, 50 mL), surgical motor, round surgical burs (0.8 mm diameter), needles, sterile saline, four cylinders, eight screws, and screwdriver ready.
  2. Preclinical treatment
    1. Acclimate the animals one week prior to surgery.
    2. Provide a prophylactic antibiotic daily (5–10 mg/kg by mouth (PO)) starting 2 h before surgery up to 3 days after surgery.
  3. Anesthesia and intubation
    1. Sedate the animals by intramuscular (IM) injection of ketamine (25 mg/kg, 50 mg/mL, 0.5 mL/kg) + xylazin (3 mg/kg, 20 mg/mL, 0.15 mL/kg). Wait ~20 min for the animals to sleep
      deeply (complete muscular atony).
      NOTE: This premedication will allow a simple, fast and painless intubation process. Deep analgesia and anesthesia is induced as described in step 2.3.8.
    2. Place an intravenous (IV) cannula into the marginal vein from the ear and keep it closed until intubation is completed.
      NOTE: This IV line will serve to perfuse fentanyl and propofol for deep analgesia and anesthesia, respectively (see step 2.3.8).
    3. Maintain anesthesia by supplying 5% sevoflurane in pure oxygen until intubation is performed.
      NOTE: This step is necessary only if the animal shows signs of awakening (eye movements, muscular contractions).
    4. Anesthetize the trachea locally by spraying 10% lidocaine. Place the rabbit in prone position and maintain its head in vertical extension.
    5. Slide the first endotracheal tube of small diameter (2.5 mm) into the rabbit trachea until airflow can be heard in the tube. This will open the larynx and facilitate the insertion of the definitive tube.
    6. Insert a guide (intubation catheter) in the tube to fix the position of the tube into the trachea. Remove the small diameter tube and slide the definitive endotracheal tube (4.9 mm) on the guide.
    7. Remove the guide and inflate the balloon at the end of the endotracheal tube to seal and block the device into the trachea. The tube will stay in place but it may be secured by using a lace tied around the forehead.  Immediately ventilate (7 mL/kg, frequency of 40/min) the animal with 3% sevoflurane in pure oxygen.
    8. Continuously perfuse (ear vein) fentanyl (0.01 mg/mL, 2–4 mL/h) to induce analgesia, 2–4 mg/kg of (2%) propofol (20 mg/mL, 4–8 mL/h) to induce anesthesia, and 4 mL/kg/h of Ringer's acetate to maintain iso-volumetric conditions.
    9. Place a rectal temperature probe. Also monitor heart function, temperature and oxygen saturation during the entire process.
    10. Control the depth of anesthesia by monitoring autonomous breathing; if the animal shows signs of autonomous breathing, dispense a small bolus of propofol and fentanyl.
  4. 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.
  5. 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.
  6. 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).
  7. 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.
  8. 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.
  9. Surgical site closure
    1. Close the skin above the cylinders with an intermittent non-resorbable suture.
    2. Apply a sprayable dressing onto the wound.

3. 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.
  5. Remove the sutures after about 10 days of wound healing.

Results

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,

Discussion

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...

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors are indebted to Geistlich AG (Wolhusen, CH) and the Osteology foundation (Lucerne, CH) (grant n°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, “experimental surgery Dpt” ,for their remarkable technical assistance.

Materials

NameCompanyCatalog NumberComments
Drugs
Enrofloxacine Baytril 10%BayerAntibiotic
FentanylBischelFor analgesia
Ketalar 50mg/mlPfizerKetamine for anesthesia
LidohexBichselLubricating gel for the eyes
OpsiteSmith and Nephew66004978Sprayable dressing
Povidone iodine 10%, BetadineMundipharmaanti-infective agent
Propofol 2%Braun3538710For anesthesia
Rapidocain 2%sinteticaLocal anesthesia
Ringer-acetateFresenius KabiVolume compensation
Rompun 2%BayerXylazin for anesthesia
Sevoflurane 5%AbbvieFor anesthesia
Sterile salineSintetica
TemgesicReckitt BenckiserBuprenorphine hydrochloride, analgesia
Thiopental InresaOspedialaFor anesthesia
Xylocaine 10% sprayAstra ZenecaFor intubation
NameCompanyCatalog NumberComments
Equipment
Fresenius Vial pilot CImexmedInfusion pump
Heated padHarvard Apparatus
Suction dominant 50Medela
Suction tubing OptimusPromedical80342.2
Surgical motorSchick dentalQubeDrilling of intramedullary holes
VentilationMaquet Servo1
NameCompanyCatalog NumberComments
Material
Cylinders and capsBoutyplastCustomizedcomposition: PEEK (poly ether ether ketone)
Manual self-retaining shaftGlobalDACT1K
Mobile handle for self-retaining shaftGlobalDMTM
Self- drilling screwsGlobalDVA1.2KL4cross-drive screws composed by Titanium grade5, ISO 5832-3
NameCompanyCatalog NumberComments
Surgical tray
Endotracheal tube Shiley diameter 2,5mmCovidien86233For intubation
Endotracheal tube Shiley diameter 4,9mmCovidien107-35GFor intubation
Ethicon prolene 4-0Ehticon8581HNon-resorbable suture
ForcepsMarcel BlancBD027R145 mm
Intubation catheterCook medicalGuide for intubation
Needlle holderMarcel BlancBM008R
Needles BD Microlance3Becton Dickinson300300/30462226G; 18G
PeriostealHU-FriedyP9X
Round surgical bursPatterson780000.8 mm in diameter, Drilling of intramedullary holes
ScalpelSwann-Mortonn°10 and n°15
ScissorsMarcel Blanc00657180 mm
Syringes OmnifixBraun4616057V5ml, 10ml and 50ml
Venflon G22Braun42690985-01Vasofix safety for the ear iv line

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