The study is supported in part by the Philanthropy Department of Mutuelles AXA through the Head and Heart Chair and the ANR BonyBrain. We acknowledge all the members of the platform SFR Necker INSERM US24, LEAT Imagine, Paris, France, for their contribution to the realization of the procedure.

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
<ol>
	<li>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).</li>
	<li>Give animals, at least 1 week before the procedure, nutritional supplement gel for acclimation.</li>
</ol>
2. Surgical procedure
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare the heating plate (37 &#176;C), place foam support on it, and fix the snout mask with small needles or adhesive tape.</li>
	<li>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.</li>
	<li>Place the anesthetized mouse on its back on the heating plate covered by a sterile field kept at 37 &#176;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.</li>
	<li>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.</li>
	<li>Perform the submandibular cutaneous approach as described below.
	<ol>
		<li>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.</li>
		<li>Dissect the subcutaneous tissues to expose the masseter muscle.</li>
	</ol>
	</li>
	<li>Disinsert the masseter muscle attaches with the use of a periosteal elevator from the inferior border of the mandible.</li>
	<li>Elevate the masseter muscle in a subperiosteal plane along the mandibular ramus with the elevator.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Perform cephalic lateral X-rays to confirm the direction of the fracture (Figure 3).
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Check the mice until they regain consciousness before placing them back in the cage.</li>
</ol>
3. Post-operative care
<ol>
	<li>Prepare a mouse cage without kibbles and hard enrichment to avoid any masticatory forces.</li>
	<li>Isolate the operated mouse in a single cage for the 1st week until the skin has completely healed. At 1 week post-operatively, group operated female mice together in the same cage.</li>
	<li>Administer buprenorphine (0.1 mg/kg) by subcutaneous injections at 4 h, 24 h, 48 h, and 72 h after the procedure.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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 &#62; 1, buprenorphine injection is given, and when the score is &#62; 3 and/or in case of weight loss &#62; 20% of preoperative weight despite food enrichment, euthanasia must be discussed (Table 1).</li>
</ol>

Standardized Mandibular Fracture Protocol for Bone Repair in Mice

<ol>
	<li>Thompson, Z., Miclau, T., Hu, D., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+intramembranous+ossification+during+fracture+healing.">A model for intramembranous ossification during fracture healing.</a> J Ortho Res. 20 (5), 1091-1098 (2002).</li><li>Bahney, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+biology+of+fracture+healing.">Cellular biology of fracture healing.</a> J Ortho Res. 37 (1), 35-50 (2019).</li><li>Yu, Y. Y., Lieu, S., Hu, D., Miclau, T., Colnot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site+specific+effects+of+zoledronic+acid+during+tibial+and+mandibular+fracture+repair.">Site specific effects of zoledronic acid during tibial and mandibular fracture repair.</a> PLoS One. 7 (2), e31771 (2012).</li><li>Landry, P. S., Marino, A. A., Sadasivan, K. K., Albright, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+soft-tissue+trauma+on+the+early+periosteal+response+of+bone+to+injury.">Effect of soft-tissue trauma on the early periosteal response of bone to injury.</a> J Trauma. 48 (3), 479-483 (2000).</li><li>Colnot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+cell+fate+decisions+within+periosteum+and+bone+marrow+during+bone+regeneration.">Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration.</a> J Bone Miner Res. 24 (2), 274-282 (2009).</li><li>Julien, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+contribution+of+skeletal+muscle+mesenchymal+progenitors+to+bone+repair.">Direct contribution of skeletal muscle mesenchymal progenitors to bone repair.</a> Nat Commun. 12 (1), 2860 (2021).</li><li>McGuire, C., Boudreau, C., Prabhu, N., Hong, P., Bezuhly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezosurgery+versus+conventional+cutting+techniques+in+craniofacial+surgery:+A+systematic+review+and+meta-analysis.">Piezosurgery versus conventional cutting techniques in craniofacial surgery: A systematic review and meta-analysis.</a> Plast Reconstr Surg. 149 (1), 183-195 (2022).</li><li>Wong, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocyte-to-osteoblast+transformation+in+mandibular+fracture+repair.">Chondrocyte-to-osteoblast transformation in mandibular fracture repair.</a> J Orthop Res. 39 (8), 1622-1632 (2021).</li><li>Vercellotti, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technological+characteristics+and+clinical+indications+of+piezoelectric+bone+surgery.">Technological characteristics and clinical indications of piezoelectric bone surgery.</a> Minerva Stomatol. 53, 207-214 (2004).</li><li>Wong, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocyte-to-osteoblast+transformation+in+mandibular+fracture+repair.">Chondrocyte-to-osteoblast transformation in mandibular fracture repair.</a> J Orthop Res. 39 (8), 1622-1632 (2021).</li><li>Tian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1&#945;+regulates+osteoclast+activation+and+mediates+osteogenesis+during+mandibular+bone+repair+via+CT-1.">HIF-1&#945; regulates osteoclast activation and mediates osteogenesis during mandibular bone repair via CT-1.</a> Oral Dis. 28 (2), 428-441 (2022).</li></ol>

The authors declare no conflicts of interest.

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 undesirable effect can abnormally increase the volume of the bone callus and delay the consolidation period. This protocol for mandibular fracture does not impact mandibular incisor growth post-surgery. To improve the effectiveness of this protocol, we advise the experimenter to first exercise on euthanized animals of similar age and dissect immediately after fracturing the mandible to directly visualize the fracture direction if necessary.
Mesenchymal progenitors migrating from adjacent muscles to the callus contribute to bone repair6. It has been demonstrated that periosteum and muscle injury impact bone consolidation5,6. In addition, extensive muscle sections lead to an increased risk of bleeding and may lead to post-operative infection. Piezosurgery has been largely developed in the field of craniofacial bone surgery; it has been reported to have lower rates of soft tissue injury and postoperative pain when compared with conventional osteotomies7,9. To minimize masseter muscle and periosteum injury, we used piezosurgery to initiate the fracture at the inferior border of the mandible and completed the fracture with scissors at the upper level of the ramus to avoid extensive masseter muscle section. This implies controlling the position of the posterior border of the mandible and the coronoid notch before fracturing the upper part of the ramus with the scissors. We encountered no difficulty in completing the fracture with the use of small straight scissors after having initiated it with piezosurgery at the lower edge of the mandible. We experienced this procedure in adult mice with different genetic backgrounds (C57BL/6J, CD1 (results not shown)), with different bone densities and mandibular sizes (prognathism, micrognathia). However, we advise checking that the length of the scissors is suitable for fracturing the upper ramus only once to avoid any abnormal fracture splits.
The risk of bleeding is minimized in this procedure due to this minimal open approach. In the event of excessive bleeding that may occur during muscular elevation, a short compression of the surgical site with gauze stops the bleeding. A potential risk of operating site infection may occur in case of molar tooth fracture when mandibular fracture is too anteriorly placed; this adverse effect can be detected on post-operative lateral X-rays. In this case, the animal must be excluded from the study. Post-operative infection of the operating site may also be due to a hematoma; this may be prevented by performing separated cutaneous sutures, allowing spontaneous evacuation when abnormal bleeding occurs preoperatively. General anesthesia with isoflurane versus&#160;intraperitoneal injection avoids excessive mortality rates linked to the adverse traumatic effects of intraperitoneal injections. In our experience, we did not observe any animal death directly linked to general anesthesia with isoflurane.
Mice may present post-operative reduction of food intake. In case of insufficient food intake or insufficient weight gain, sifted kibble powder may be added to the gel. To improve postoperative food intake, we recommend giving nutritional supplement gel with kibbles at least 1 week before the procedure for accommodation. We recommend performing additional X-rays during the follow-up (every week, during the follow-up) and at the time of the sacrifice to evaluate potential fracture displacement. In non-pathological conditions, bone consolidation is complete 4 weeks after the fracture.
Several authors have described different protocols for non-stabilized fractures of the mandible in mice3,10,11. The instruments used to perform the osteotomy can be different, namely, drills, surgical tweezers, etc. However, the use of drills may lead to larger bone loss and soft tissue injuries. To the best of our knowledge, this procedure of non-stabilized mandibular fractures is the first to associate piezosurgery and scissors to complete the osteotomy. This technique has the advantage of preserving soft tissues while permitting a precise osteotomy with minimal bone loss.
As for all techniques, this method has some limitations. We preferred performing the fracture with the use of scissors after initiating it with a piezoelectric surgery insert to avoid any extended masseter muscle section and periosteum injury that may exacerbate a potential consolidation delay and/or formation of pseudarthrosis. However, this technique implies controlling the position of the coronoid notch without total visualization of the mandibular ramus to perform the fracture within the same direction. A learning curve is thus expected, depending on the surgical experience. In addition, the direction of the mandibular fracture on X-rays may be difficult to visualize depending on the X-ray device. We did not use in vivo micro-CT to confirm the fracture direction, as it could compromise mouse recovery because of the increased duration of general anesthesia.
Studying the endochondral bone repair process implies the realization of non-stabilized fractures, thus allowing a better understanding of the bone repair process. This step is necessary if we want to consider preclinical trials in mouse models with bone repair defects with the aim of developing new clinical applications for treating defective bone repair and pseudarthrosis in patients in the context of genetic diseases or in the case of post-traumatic or post-tumoral sequelae.

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 site4,5,6.
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 models3,7, 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.

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

less-invasive technique for non-stabilized mandibular fracture in mouse models

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.

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 performed, deparaffined in deparaffinization solution, and then rehydrated prior to Sirus Red/Alcian Blue staining, using standard protocols. The histological images confirmed the formation of a soft callus with a cartilaginous template (Figure 5). Bone healing is completed at 4 weeks after the fracture, as shown by micro-CT analyses. We suggest studying the bone repair process at different key points from the onset of callus formation (e.g., every 7 days (day 7, 14, 21) or more depending on the aim of the study) to the end of bone healing (i.e., day 28), with histomorphological and micro-CT morphometric analyses.
Based on a retrospective analysis of this procedure performed on 58 wild-type mice (C57BL/6J background), the success rate of the protocol is 86.3% and an exclusion rate of 13.7%. Exclusion was due to premature spontaneous death in the early postoperative period (3%), to the decision of euthanasia for mice presenting a critical score (1%; score &#62; 3 and/or in case of weight loss &#62; 20%), or in case of abnormal position of the osteotomy, with abnormal split of the mandibular angle (5%), or when the fracture was at the molar level (n=1/58). Infection was observed in the latter case, with the abnormal splitting of the fracture at the molar level (n= 1/58; Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66824/66824fig01.jpg" /> 
Figure 1: Image of the submandibular cutaneous approach of the mandibular ascending branch. The inferior border of the mandible is fractured with a piezotome insert, allowing minimal bone loss (arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66824/66824fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66824/66824fig02.jpg" /> 
Figure 2: Staining of mouse mandible. Lateral view of a 6-week-old wild type mouse mandible (stained with alizarin red / alcian blue staining): 1. Condyle, 2. Coronoid process, 3. Coronoid notch, 4. Angle process, 5. Posterior border of the ramus, 6. Basilar (or inferior) border of the mandible. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66824/66824fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66824/66824fig03.jpg" /> 
Figure 3: X-ray of the mouse skull. Lateral X-rays of the skull of a wild type 6-week-old mouse (sagittal view) showing the aspect of the mandible of an unfractured mouse (left) and the aspect of the mandible of a fractured mouse and the direction of the non-stabilized mandibular fracture (arrows), from the inferior border to the coronoid notch, placed anteriorly to the condyle (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66824/66824fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66824/66824fig04.jpg" /> 
Figure 4. X-ray of the mouse skull under suboptimal conditions. Lateral X-rays of the skull of a wild-type 6-week-old mouse (sagittal view) showing abnormal fracture of the mandibular angle (arrows). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66824/66824fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66824/66824fig05.jpg" /> 
Figure 5: Representative results. Results at day 14 post-fracture in a wild-type mouse (C57BL/6J). Representative 3D coronal views of a reconstructed &#181;CT scan of the mandibular fracture site, showing the bone callus (white arrows). Representative images of the callus cartilage (Sirus Red/Alcian Blue staining) confirming the presence of an endochondral bone repair process. Abbreviations: B = Bone, C = Cartilage. Scale bar: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66824/66824fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>0</td>
			<td>1</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Animal aspect</td>
			<td>Normal</td>
			<td>Toilet reduction</td>
			<td>Ruffled hairs, arched back</td>
		</tr>
		<tr>
			<td>Behavior</td>
			<td>Normal</td>
			<td>Hypomobility</td>
			<td>Hypomobility, reduction of food intake, weight loss &#62; 10 %</td>
		</tr>
		<tr>
			<td>Clinical signs</td>
			<td>Normal</td>
			<td>Scar inflammation</td>
			<td>Opened scar, infection</td>
		</tr>
	</tbody>
</table>
Table 1. Post-operative well-being score scale. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>n = 58 mice</td>
		</tr>
		<tr>
			<td>Success rate</td>
			<td>86.3 %</td>
		</tr>
		<tr>
			<td>Exclusion rate :</td>
			<td>13.7 %</td>
		</tr>
		<tr>
			<td>Premature spontaneous death</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>Critical score &#60; 3, and/or weight loss &#62; 20 %</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Abnormal fracture split to the mandibular angle</td>
			<td>5%</td>
		</tr>
		<tr>
			<td>Abnormal fracture split, at molar level</td>
			<td>1.7 %</td>
		</tr>
	</tbody>
</table>
Table 2. Success and exclusion rates of the protocol.

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

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.

Non-stabilized fractures can be made at mandibular sites in mice, thus making it possible to analyze bone repair using an endochondral ossification mode. ...

less-invasive-technique-for-non-stabilized-mandibular-fracture-mouse

Nanyang Technological University

Research

JoVE Journal

Biology

250 Views.  Université de Paris Cité. 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. 

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

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Medicine

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Neuroscience

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

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

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Chapters in this video

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

A Simple Critical-sized Femoral Defect Model in Mice

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Establishing a Diaphyseal Femur Fracture Model in Mice