Less-Invasive Technique for Non-stabilized Mandibular Fracture in Mouse Models

Podsumowanie

This protocol presents a comprehensive and efficient method for analyzing mandibular bone repair. We describe a reproducible technique for non-stabilized mandibular fracture in a mouse model, allowing analysis of the process of endochondral bone repair with minimal tissue damage and bone loss.

Streszczenie

Non-stabilized fractures can be made at mandibular sites in mice, thus making it possible to analyze bone repair using an endochondral ossification mode. To most accurately reflect this process in vivo, it is necessary to have a standardized protocol to avoid excessive bone loss and soft tissue damage, particularly at the mandibular site, an anatomical site characterized by minimal access. To our knowledge, we describe for the first time a less-invasive protocol of non-stabilized mandibular fracture in mice. Adult mice are anesthetized with isoflurane and receive a preoperative dose of buprenorphine by subcutaneous injection. A submandibular approach is performed, with a skin incision made along the inferior border of the mandible. The masseter muscle is elevated in a subperiosteal plane along the mandibular ramus with a periosteal elevator. A vertical and complete fracture of the ramus is performed from the basilar border to the coronoid notch (between condylar and coronoid processes), using a piezoelectric bone surgery device under saline serum irrigation at the basilar edge and scissors to complete the fracture of the mandibular ramus. The skin approach is closed by silk sutures. This detailed procedure for non-stabilized mandibular fractures offers an efficient procedure allowing minimal bone loss and soft tissue damage. This technique ensures successful and consistent results to accurately reflect the process of endochondral bone repair in mouse models.

Wprowadzenie

Bone repair implies both membranous and endochondral ossification processes, depending on the fracture or osteotomy stabilization. Bone repair following stabilized bone osteotomies or fractures relies on membranous ossification, whereas non-stabilized fractures or poorly stabilized osteotomies consolidate according to a mode of endochondral ossification¹. Membranous bone repair is characterized by bone formation by direct differentiation of mesenchymal cells into osteoblasts, whereas endochondral bone repair involves the formation of a transient cartilaginous template, which differentiates into osteoblasts to form the bone callus².

Bone repair depends on multiple tissue and cellular interactions; it is also influenced by site-specific factors and by the force exerted on the callus³. Mandibular bone is a mobile bone supporting masticatory forces, mostly formed by cortical bone. The process of mandibular bone repair is influenced by the site and the direction of the fracture. It is well known that muscular and periosteum injuries may impact bone consolidation as well as important bone loss at fracture site^4,5,6.

Analyzing endochondral mandibular bone repair implies standardized and reproducible protocols for valuable, non-biased comparisons. Regarding literature, only a few authors report protocols for non-stabilized mandibular fractures in mice models^3,7, and mostly with an anterior direction at molar level⁸. By developing this technique, we aim to avoid muscular and periosteum injuries and limit bone loss at the fracture site to best analyze the process of endochondral bone repair without confounding factors. In addition, the purpose of developing a new technique for mandibular fracture in mice was to minimize post-operative pain and animal death frequent in such surgery in small animals. For the first time, we describe a less-invasive protocol for non-stabilized mandibular fracture at the ramus level in mice. This in vivo protocol avoids excessive muscular and periosteum damage or bone loss at the fracture site and helps reduce the level of postoperative pain and the risk of death of the animal.

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Protokół

This protocol follows the animal care guidelines of Imagine Institute and has been validated by ethical committees and the French ministry (APAFIS 26995).

1. Animals

1. Determine the number of animals per group. We recommend 12 animals per group. Determine the sex and age of mice. This protocol is adapted for 6-week-old C57BL/6J mice (male or female).
2. Give animals, at least 1 week before the procedure, nutritional supplement gel for acclimation.

2. Surgical procedure

1. Prepare the material required. Autoclave the instruments: scissors, needle driver, forceps, insert and handpiece of the piezoelectric bone surgery device, and elevator. Prepare sterile gloves, silk sutures (4/0), shaver, cotton gauze, 10% povidone-iodine solution, heating table, isoflurane, and the snout mask for isoflurane inhalation.
2. Prepare buprenorphine at 0.1 mg/kg, diluted in sterile injectable physiological serum. Administer buprenorphine by subcutaneous injection with a 25G needle 30 min before surgery.
3. Prepare the heating plate (37 °C), place foam support on it, and fix the snout mask with small needles or adhesive tape.
4. Place the mouse in the isoflurane tank for induction, with isoflurane diffusion (4%). Test anesthesia depth by performing a toe pinch. In case of sufficient induction, a lack of pedal reflex will be observed.
5. Place the anesthetized mouse on its back on the heating plate covered by a sterile field kept at 37 °C during the entire procedure. Position the mask on the snout for continuous isoflurane inhalation (maintenance at 2.5%). Fix the mouse extremities with adhesive tape.
6. Apply eye lubricant bilaterally to prevent eye dryness induced by general anesthesia. Shave the submandibular hair, wear sterile gloves, and disinfect the skin with several swipes of 10% povidone-iodine solution and alcohol.
7. Perform the submandibular cutaneous approach as described below.
   1. Perform a skin incision from the mandibular angle to the 1/3 posterior - 2/3 anterior junction of the horizontal branch of the mandible with micro scissors.
   2. Dissect the subcutaneous tissues to expose the masseter muscle.
8. Disinsert the masseter muscle attaches with the use of a periosteal elevator from the inferior border of the mandible.
9. Elevate the masseter muscle in a subperiosteal plane along the mandibular ramus with the elevator.
10. Create a small osteotomy (3 mm length) at the inferior border of the mandible using the Piezoelectric bone surgery device under saline serum irrigation (10 mL/min maximum) at the inferior border of the mandible, anteriorly to the mandibular angle, in the posterior part of the concavity (Figure 1, Figure 2).
11. Locate the posterior border of the mandible and the coronoid notch (with the micro scissors or the forceps) and complete the fracture with the use of small straight scissors inserted along the mandibular ramus to avoid additional masseter muscle aperture and injury. To allow correct orientation of the fracture, place the scissors perpendicularly to the inferior border of the mandible.
12. Verify that the fracture is totally complete: total mobility between the two bone segments should be observed during soft mobilization of the 2 segments with forceps.
13. Close the skin approach with silk sutures (4/0) with separated sutures. No suture is needed in the periosteum.
    NOTE: Alternatively, the skin approach may be sutured with other non-adsorbable sutures (e.g., Polypropylene sutures).
14. Perform cephalic lateral X-rays to confirm the direction of the fracture (Figure 3).
    1. Place the mouse again in the isoflurane tank for short reinduction before performing the X-rays. Then, place the mouse in extension, on the lateral side, with the head slightly in hyperextension for X-ray analysis.
    2. In case of fracture too anteriorly placed at the level of a mandibular molar or when the fracture detached the mandibular angle (Figure 4), observe an abnormal increase in the volume of the bone callus. In these cases, exclude the animals from the study to avoid any bias in analysis.
15. Check the mice until they regain consciousness before placing them back in the cage.

3. Post-operative care

1. Prepare a mouse cage without kibbles and hard enrichment to avoid any masticatory forces.
2. Isolate the operated mouse in a single cage for the 1^st week until the skin has completely healed. At 1 week post-operatively, group operated female mice together in the same cage.
3. Administer buprenorphine (0.1 mg/kg) by subcutaneous injections at 4 h, 24 h, 48 h, and 72 h after the procedure.
4. Feed mice with soft diet gel during all post-operative periods from day 0 to the end of the consolidation period (i.e., 28 days) to avoid pain and secondary displacement of the fracture.
5. Daily weigh the mice from day 0 to day 4 post fracture, in the morning, before buprenorphine injection, and then 3x per week until animal sacrifice (14 days post-fracture).
6. Evaluate post-operative animal well-being to optimize post-operative animal care. Use the score scale in Table 1 to evaluate the postoperative state of the animal and adapt the follow-up. When the score is > 1, buprenorphine injection is given, and when the score is > 3 and/or in case of weight loss > 20% of preoperative weight despite food enrichment, euthanasia must be discussed (Table 1).

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Wyniki

As for in vivo surgical procedures, to optimally perform this procedure of non-stabilized mandibular fracture in a mouse model, specific learning and training are required.

This technique for non-stabilized mandibular fracture allows the study of the endochondral bone repair process. Following euthanasia, mandibular samples were collected in 4% paraformaldehyde (24 h). After full decalcification with EDTA pH 8, sections were embedded in paraffin. Sagittal serial sections were performe...

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Dyskusje

The technique allows the realization of non-stabilized mandibular fractures in a mouse model within a minimal open approach in a short procedure time (around 10 min or less). This short-time procedure limits the risk of animal morbidity and death, especially in small animals such as mice. To obtain comparable results in the analysis of the endochondral bone repair process, particular care should be taken to perform the mandibular fractures in the same direction without abnormal split of the mandibular angle, as this unde...

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Materiały

Name	Company	Catalog Number	Comments
Buprenorphine : Buprecare 0.3 mg/kg	Animalcare
Elevator OBWEGESER width 6mm Length 17,5cm	Collin Medical	HA 5905
Forceps : Pad Plate MORIA 11 cm N°5	MORIA	9906
Heating table Bioseblab 55 cm x 33 cm	Bioseblab	707
Needle HOLDER : Micro Halsey Needle Holder - Metal	LABODERM	21,100
Neo Clear	Merck Millipore, Darmstadt, Germany	109843
Ocry Gel Tube 10 g (eye lubricant)	tvm lab	3.70045E+12
Piezotome 2 ASS FINAL	ACTEON	X57402
Piezotome insert Piezocision PZ3	ACTEON	F87574
Scissors micro	MORIA	MC52
Silk sutures PERMA HAND SEIDE  / MERSILK	ETHICON	REF 18501G
Straight scissors	MORIA	4877A
Vetflurane (isoflurane) 250 ml	VIRBAC	VET066 (Centravet)