Name: transverse fracture of the mouse femur with stabilizing pin
Uploaded: 2021-12-29T13:30:00
Description: Watch this Scientific Journal Video about Transverse Fracture of the Mouse Femur with Stabilizing Pin at JoVE.com

We thank Dr. Vicki Rosen for financial support and guidance with the project. We would also like to thank the veterinary and IACUC staff at the Harvard School of Medicine for consultation regarding sterile technique, animal well-being, and the materials used to develop this protocol.

All animal experiments described were approved by the Institutional Animal Care and Use Committee of the Harvard Medical Area. 12-week-old C57BL/6J mice (males and females) were used in this protocol. C57BL/6J male and female mice achieve peak bone mass around 12 weeks of age with femurs wide enough to fit a stabilizing pin, making them an appropriate strain to use for this protocol15.
1. Preparation for the surgery
<ol>
	<li>Autoclave the surgical eq...

<ol>
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The injury model detailed in this protocol encompasses all four significant steps observed during the healing of spontaneous fractures, including (1) pro-inflammatory response with the formation of the hematoma, (2) recruitment of skeletal progenitors from the periosteum to form the soft callus, (3) mineralization of the callus by osteoblasts and (4) remodeling of the bone by osteoclasts.
The surgical procedure described in this manuscript is optimized for adult mice at least 12 weeks old. A 2...

Fractures, breaks in the continuity of the bone surface, occur in all segments of the population. They become severe in people who have fragile bones due to aging or disease, and the health care costs of fragility fractures are expected to exceed $25 billion in 5 years1,2,3,4,5. Understanding the biological mechanisms involved in fracture repair would be a starting point in developing new therapies aimed at enhancing the healing process. Previous research has shown that, upon fracture, four significant steps occur that enable bone to heal: (1) formation of the hematoma; (2) formation of a fibrocartilaginous callus; (3) mineralization of the soft callus to form bone; and (4) remodeling of the healed bone6,7. Many biological processes are activated to heal the fracture successfully. First, an acute pro-inflammatory response is initiated immediately after a fracture6,7. Then, the periosteum becomes activated and expands, and periosteal cells differentiate into chondrocytes to form a cartilage callus that grows to fill the gap left by the disrupted bone segments6,7,8,9. Neural and vascular cells invade the newly formed callus to provide additional cells and signaling molecules needed to facilitate repair6,7,8,9,10. In addition to contributing to callus formation, periosteal cells also differentiate into osteoblasts that lay down woven bone in the bridging callus. Finally, osteoclasts remodel the newly formed bone to return to its original shape and lamellar structure7,8,9,10,11. Many groups developed mouse models of fracture repair. One of the earlier and most often used fracture models in mice is the Einhorn approach, where a weight is dropped on the leg from a specific height12. The lack of control over the angle and the force applied to induce the fracture creates a lot of variability in the location and size of the bone discontinuity. Subsequently, it results in variations in the specific fracture healing response observed. Other popular approaches are surgical intervention to produce a tibial monocortical defect or stress fractures, procedures that induce comparatively milder healing responses10,13. Variability in these models is primarily due to the person conducting the procedure14.
Here, a detailed mouse femur injury model allows for control over the break to provide a reproducible injury and allow for quantitative and qualitative assessment of femur fracture repair. Specifically, a complete breakthrough in the femurs of adult mice is introduced and stabilizes the fracture ends to account for the role physical loading plays in bone healing. The methods for harvesting tissues and imaging the different steps of the healing process using histology and microcomputed tomography (microCT) are also provided in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>23 G x 1 TW IM (0.6 mm x 2 5mm) needle</td>
			<td>BD precision</td>
			<td>305193</td>
			<td>Use as guide needle</td>
		</tr>
		<tr>
			<td>27 G x 1 &#188; (0.4 mm x 30 mm)</td>
			<td>BD precision</td>
			<td>305136</td>
			<td>Use as stabilizing pin</td>
		</tr>
		<tr>
			<td>9 mm wound autoclip applier/remover/clips kit</td>
			<td>Braintree Scientific, INC</td>
			<td>ACS-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8 GX</td>
			<td>Electron Microscopy Sciences</td>
			<td>10350</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Millipore Sigma</td>
			<td>AX1303</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular blade X926.7 THIN-FLEX</td>
			<td>Abrasive technologies</td>
			<td>CELBTFSG633</td>
			<td></td>
		</tr>
		<tr>
			<td>DREMEL 7700-1/15, 7.2 V Rotary Tool Kit</td>
			<td>Dremel</td>
			<td>7700 1/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>ThermoScientific</td>
			<td>7111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine curved dissecting forceps</td>
			<td>VWR</td>
			<td>82027-406</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxulin Gill 2</td>
			<td>Sigma-Aldrich</td>
			<td>GHS216</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Millipore Sigma</td>
			<td>HX0603-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical kit</td>
			<td>VWR</td>
			<td>95042-540</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma-Aldrich</td>
			<td>1625</td>
			<td></td>
		</tr>
		<tr>
			<td>Phloxine B</td>
			<td>Sigma-Aldrich</td>
			<td>P4030</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine Swabs</td>
			<td>PDI</td>
			<td>S23125</td>
			<td></td>
		</tr>
		<tr>
			<td>SCANCO Medical &#181;CT35</td>
			<td>Scanco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slow-release buprenorphine</td>
			<td>Zoopharm</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In C57BL/6J mice, a successful surgery completes the healing steps mentioned earlier with little to no local inflammatory response or periosteal involvement in the sham-operated contralateral femur. A hematoma is formed a few hours after surgery, and the periosteum is activated to recruit skeletal progenitors for chondrogenesis. Various cell populations, such as Prx1+ mesenchymal progenitors, can be traced during the repair process using commercially available fluorescent reporter mouse models (<strong class="...

Watch this Scientific Journal Video about Transverse Fracture of the Mouse Femur with Stabilizing Pin at JoVE.com

Transverse Fracture of the Mouse Femur with Stabilizing Pin

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 ...

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