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 equipment, including surgical scissors, straight forceps, curved forceps, surgical clamps, and diamond cutting wheel (see Table of Materials) to minimize the risk of infection.</li>
	<li>Place a clean mouse cage on a heating pad to facilitate post-surgical recovery. Set the heat pad to reach a temperature between 37-45 &#176;C.</li>
	<li>Place the mouse under anesthesia using an isoflurane chamber. Set the induction oxygen flow at 2 L/min, the isoflurane induction at 2-4%, the maintenance nose cone oxygen at 2 L/min, and the maintenance isoflurane is 1.4%.</li>
	<li>Confirm that the mouse&#39;s breathing is stable and they do not respond to a toe pinch. Apply a thin layer of ophthalmic ointment on each eye to prevent corneal scratching. Transfer the mouse to a sterile pad and maintain anesthesia using a nose cone to deliver isoflurane continuously at the same rate as in step 1.3. 
	NOTE: Using 12-week-old mice or older is recommended as femurs from younger mice might be too thin to accommodate a stabilizing pin.</li>
	<li>Inject the mouse subcutaneously with 0.05 mg/kg body weight of slow-release buprenorphine (see Table of Materials).</li>
	<li>Using an electric trimmer, shave a 2 x 2 cm square on both thighs corresponding to the location of the femur.</li>
	<li>Disinfect the shaved area using sterile gauze or swabs to spread a layer of iodine followed by a rinse with 70% ethanol (Figure 1A).</li>
</ol>
2. Surgery
<ol>
	<li>Using a sterile scalpel, make a 5 mm incision in the shaved, disinfected region and peel away the skin to expose the underlying fascia.</li>
	<li>Use straight forceps and fine scissors to delicately grab and cut the fascia directly covering the femur to expose the muscle. A 5 mm cut of the fascia is sufficient to access the underlying muscle.</li>
	<li>Using one pair of straight forceps, gently separate the muscle from the femur with minimal tissue damage.</li>
	<li>Once the femur is visible, slide the curved forceps underneath the femur between the separated muscle and bone. Let the forceps slowly open to maintain muscle separation and secure the femur to facilitate a clean cut. 
	NOTE: The femur must stay exposed and separated from the muscle and skin when the forceps are not held, as shown in Figure 1B.</li>
	<li>Make a transverse cut in the middle of the femur shaft using a handheld saw on the low power setting (See Table of Materials for the blade and Rotary Tool Kit used). 
	NOTE: Once the femur is completely cut, two fracture ends are created: the proximal section (attached to the hip bone) and the distal section (attached to the knee, also known as the stifle joint). Avoid cutting the femur in one single motion. Instead, make 3-5 passes until the femur is completely cut through. This is critical to avoid overheating the surrounding tissue and generating significant bone debris, negatively impacting healing.</li>
	<li>Insert a guide needle (23 G x 1 TW IM, 0.6 mm x 25 mm) (see Table of Materials) in the marrow cavity of the distal section. Use fingers to gently twist the needle as you push to thread through the knee joint (Figure 1C).
	<ol>
		<li>Remove the guide needle from the distal end and repeat on the proximal end, using the same gentle twisting motion to push the guide needle through the hip joint. Leave the guide needle in the proximal end, with its tip emerged from the skin (Figure 1D). 
		NOTE: To stabilize the fracture ends, a guide needle was first used to create a pathway through the fracture ends, and then a stabilizing pin was threaded through this pathway to secure the fracture ends14.</li>
	</ol>
	</li>
	<li>Insert the stabilizing pin (needle, 27 G x 1 &#188;, 0.4 mm x 30 mm) (see Table of Materials) into the tip of the guide needle (Figure 1E). Gently push so that the stabilizing pin enters as the guide needle exits the marrow cavity of the proximal end.
	<ol>
		<li>Discard the guide needle. Use tweezers to hold and align the distal end with the proximal end and continue to thread the stabilizing pin through the distal marrow cavity until it exits the knee joint using the pathway made in 2.6 (Figure 1F). 
		NOTE: The stabilizing pin should now be protruding from the hip and the knee joints.</li>
	</ol>
	</li>
	<li>Using the surgical clamp, pull the tip of the pin to bring the proximal and distal sections close together, so they are barely touching. Realign the fracture ends with forceps if needed, and fold the ends of the stabilizing pin toward the fracture site using the surgical clamp (see Table of Materials).
	<ol>
		<li>Remove the plastic from the base of the needle using wire cutters. Using the clamp, twist both ends of the pin until they are blunt to avoid internal tissue damage. 
		NOTE: The fracture ends are now locked in place so that the mouse can put weight on the injured leg. The pin is secured if the fracture ends are unable to separate. A wire cutter can also be used to blunt the ends. The stabilizing pin must stay in place for the whole duration of the study until the mouse is euthanized. Attempts at dislodging the pin are likely to destabilize the fracture response and cause harm to the animal. The pin can be removed at dissection.</li>
	</ol>
	</li>
	<li>Use straight forceps to reposition the muscle onto the femur. Using curved forceps, pinch the ends of the skin together and close the opening using wound clips. 
	NOTE: Do not close the skin too tightly with the clips or the mouse will avoid putting weight on this leg. Limiting physical loading during recovery could delay the healing process.</li>
	<li>Repeat steps 2.2 to 2.4 on the other leg and close the wound without performing the femur fracture. 
	NOTE: This sham-operated femur serves as the contralateral control.</li>
	<li>Withdraw the isoflurane exposure, place the mouse in the heated cage, and ensure they regain consciousness within 10-15 min.</li>
	<li>Monitor the mouse&#39;s activity and incision site daily for 5 days after surgery for signs of distress or infection. 
	NOTE: If the animal demonstrates signs of pain, is not eating, and/or hesitates to walk on their hindlimbs, consult with veterinary personnel and administer additional analgesic.</li>
	<li>Remove the wound clips 10 days after the surgery. 
	&#8203;NOTE: If the wound does not appear to have closed after 10 days, consult the veterinary personel and IACUC before removing the clips.</li>
</ol>
3. Tissue harvest
<ol>
	<li>Euthanize the mice via CO2 inhalation followed by cervical dislocation. 
	NOTE: This procedure is consistent with the Panel on Euthanasia of the American Veterinary Medical Association.</li>
	<li>Using scissors and forceps, remove the skin from both mouse legs, dislocate the femoral head from the hip bone, and cut adjacent muscle to free the leg. 
	NOTE: Avoid removing too much muscle around the femur as the callus could be dislodged or damaged.</li>
	<li>Avoiding the stabilizing pin, cut through the knee joint to separate the femur from the tibia.</li>
	<li>Dissect around the distal and proximal parts of the femur to expose the ends of the stabilizing pin.</li>
	<li>With a hard wire cutter, cut the folded blunt ends of the pin so only the straight portion of the pin remains. Use forceps to slowly and gently slide the stabilizing pin out of the femur. 
	&#8203;NOTE: If the pin does not easily slide out, do not apply force as this could dislodge the callus and damage the sample. Instead, try to rotate the pin and remove it delicately. Removal of the pin may also be easier after fixation.</li>
</ol>
4. Histology - Alcian Blue / Eosin /Orange G staining
NOTE: Alcian Blue/Orange G/Eosin staining is routinely used to visualize cartilage (blue) and the bone (pink). The cartilage area can be quantified as a proportion of the total callus area (Figure 2A,B).
<ol>
	<li>Fix femurs in 10% neutral buffered formalin overnight at 4 &#176;C.</li>
	<li>Wash the fixed samples in phosphate-buffered saline (PBS).</li>
	<li>Place samples in 0.5M EDTA, pH 8.0 on a slowly rotating shaker for 2 weeks. Change EDTA solution every other day to ensure efficient decalcification. 
	NOTE: No specific rpm is required for the shaker; ensure that the liquid flows in the container to cover all the samples. Complete decalcification can be tested by X-ray of the femurs.</li>
	<li>To process the samples for paraffin embedding incubate them in the following solutions (1 h each): 70% EtOH, 95% EtOH, 100% EtOH, 100% EtOH, Xylene, Xylene, Paraffin, Paraffin.</li>
	<li>Embed the samples in paraffin for sectioning.</li>
	<li>Cut 5-7 &#956;m thick longitudinal sections of the fractured femurs using a microtome.</li>
	<li>Deparaffinize the sections by incubating them in 2 baths of Xylene (5 min each).</li>
	<li>Rehydrate the sections by incubating in the following ethanol gradient (2 min each): 100% EtOH, 100% EtOH, 80% EtOH, 70% EtOH.</li>
	<li>Place slides in tap water for 1min.</li>
	<li>Make the solutions of Alcian blue, Acid alcohol, Ammonium water, and Eosin/Orange G for histology as mentioned in Supplementary File 1. 
	NOTE: The suggested volumes of each solution should be sufficient to completely submerge the slides for the staining.</li>
	<li>Place slides in Acid alcohol for 30 s and incubate in Alcian blue for 40 min.</li>
	<li>Wash gently under the running tap until the water is clear for about 2 min.</li>
	<li>Dip the slides rapidly in Acid alcohol for 1 s.</li>
	<li>Rinse as described in step 4.9.</li>
	<li>Incubate in ammonium water for 15 s.</li>
	<li>Rinse as described in step 4.9.</li>
	<li>Place in 95% EtOHfor 1 min and incubate in Eosin/Orange G for 90 s.</li>
	<li>Dehydrate the slides quickly with a single dip in 70% EtOH, 80% EtOH and 100% EtOH.</li>
	<li>Place slides in Xylene for 1 min to clear the slides.</li>
	<li>Apply mounting media and a coverslip for imaging. 
	&#8203;NOTE: For molecular analysis, RNA and protein can be isolated from the callus. Carefully dissect the muscle under a dissecting scope and separate the callus from the underlying bone using a scalpel.</li>
</ol>
5. MicroCT
NOTE: In the later stages of healing, microCT can be performed to image and quantify the mineralization in the hard callus and the fracture gap. In C57BL/6J mice, the callus is usually mineralized and detectable by microCT after 10 days post-fracture (dpf) (Figure 2C).
<ol>
	<li>Fix femurs in 10% neutral buffered formalin overnight at 4 &#176;C. 
	NOTE : MicroCT can be performed on the same femurs used for histology as long as it is done before EDTA decalcification (step 4.3). When using the same femurs for both techniques, perform microCT, retrieve the samples and go to step 4.3.</li>
	<li>Wash the fixed samples in phosphate-buffered saline (PBS) and store in 70% EtOH.</li>
	<li>Perform microCT with an isotropic voxel size of 7 &#956;m at an energy level of 55 kVP and an intensity of 145 &#956;A (see Table of Materials).</li>
	<li>Contour the microCT slices to include the callus and exclude the cortical bone. 
	NOTE: As the callus gets more mineralized with time, the threshold can be adjusted to visualize and measure the callus volume at different stages.</li>
	<li>Obtain the bone volume contained in the callus contours as a measurement of the callus volume. 
	NOTE: Fracture gap can be measured directly on the microCT slices as the distance occupied by the break.</li>
</ol>

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<li>Curtis, E. M., Moon, R. J., Harvey, N. C., Cooper, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+impact+of+fragility+fracture+and+approaches+to+osteoporosis+risk+assessment+worldwide%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">The impact of fragility fracture and approaches to osteoporosis risk assessment worldwide.</a> Bone. 104, 29-38 (2017).</li>
<li>Laurent, M. R., Dedeyne, L., Dupont, J., Mellaerts, B., Dejaeger, M., Gielen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+bone+loss+and+sarcopenia+in+men%5BTitle%5D)+AND+%22Maturitas%22%5BJournal%5D)">Age-related bone loss and sarcopenia in men.</a> Maturitas. 122, 51-56 (2019).</li>
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<li>Williams, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Economic+burden+of+osteoporotic+fractures+in+US+managed+care+enrollees%5BTitle%5D)+AND+%22The+American+Journal+of+Managed+Care%22%5BJournal%5D)">Economic burden of osteoporotic fractures in US managed care enrollees.</a> The American Journal of Managed Care. 26, 142-149 (2020).</li>
<li>Sheen, J. R., Garla, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fracture+healing+overview%5BTitle%5D)+AND+%22StatPearls%22%5BJournal%5D)">Fracture healing overview.</a> StatPearls. , https://www.ncbi.nlm.nih.gov/books/NBK551678 (2021).</li>
<li>Holmes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Closing+the+gap%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Closing the gap.</a> Nature. 550, 194-195 (2017).</li>
<li>Duchamp de Lageneste, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Periosteum+contains+skeletal+stem+cells+with+high+bone+regenerative+potential+controlled+by+Periostin%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin.</a> Nature Communications. 9, 773(2018).</li>
<li>Bahney, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+biology+of+fracture+healing%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Cellular biology of fracture healing.</a> Journal of Orthopaedic Research. 37, 35-50 (2019).</li>
<li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fracture+repair+requires+TrkA+signaling+by+skeletal+sensory+nerves%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Fracture repair requires TrkA signaling by skeletal sensory nerves.</a> Journal of Clinical Investigation. 129, 5137-5150 (2019).</li>
<li>Colnot, C., Thompson, Z., Miclau, T., Werb, Z., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+fracture+repair+in+the+absence+of+MMP9%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Altered fracture repair in the absence of MMP9.</a> Development. 130, 4123-4133 (2003).</li>
<li>Bonnarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+a+standard+closed+fracture+in+laboratory+animal+bone%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Production of a standard closed fracture in laboratory animal bone.</a> Journal of Orthopaedic Research. 2, 97-101 (1984).</li>
<li>Hu, K., Olsen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoblast-derived+VEGF+regulates+osteoblast+differentiation+and+bone+formation+during+bone+repair%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair.</a> Journal of Clinical Investigation. 126, 509-526 (2016).</li>
<li>Collier, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+reproducible+model+of+fracture+healing+in+mice+using+an+open+femoral+osteotomy%5BTitle%5D)+AND+%22Bone+Reports%22%5BJournal%5D)">Characterization of a reproducible model of fracture healing in mice using an open femoral osteotomy.</a> Bone Reports. 12, 100250(2020).</li>
<li>Glatt, V., Canalis, E., Stadmeyer, L., Bouxsein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+changes+in+trabecular+architecture+differ+in+female+and+male+C57BL%2F6J+mice%5BTitle%5D)+AND+%22Journal+of+Bone+and+Mineral+Research%22%5BJournal%5D)">Age-related changes in trabecular architecture differ in female and male C57BL/6J mice.</a> Journal of Bone and Mineral Research. 22, 1197-1207 (2007).</li>
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The authors do not have any conflicts of interest to disclose.

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 27 G x 1 &#188; (0.4 mm x 30 mm) needle is used as the stabilizing pin because it is the ideal size for the width of the marrow cavity at this age. If needed, the protocol could be amended for younger animals if a thinner stabilizing pin is used. The stabilizing pin is an essential part of the surgery&#39;s success since instability is known to significantly affect fracture healing16. Other stabilization methods have been reviewed and all come with their advantages and limitations17,18,19. The ideal stabilization should be selected based on the research question and experimental goal. One limitation of the described stabilization here is that the pin goes through the growth plates and the joints. If the contribution from the articular or growth plate cartilage is of concern, we suggest considering another stabilizing method.
Variability in surgical technique and between animals is a concern when performing this surgery. Therefore, it is recommended to use ample numbers, especially for quantitative readouts, and to compare similar types of fractures across groups. It is critical to secure the femur sections close together to avoid gaps over 2 mm. Variability in gaps between sections can considerably affect the size of the callus and the timing of repair. The sections should also be properly aligned. Loose and misaligned fractures will cause more significant variability across samples and might impair healing.
Weight-bearing is also crucial for the timing of bone healing and can introduce variability between mice. Most mice put minimal weight on the injured leg a few hours after surgery but should walk normally by the next day. Verifying that the mouse moves normally and the load is distributed evenly on both legs is crucial, especially when using the contralateral femur as a sham-operated control. In addition, the surgery can trigger a systemic inflammation that affects the contralateral leg. It is therefore recommended to compare sham-operated femurs to non-operated mice when establishing this technique. Having non-operated controls could also be preferable for quantifiable outcomes such as effects on RNA or protein expression.
Consistency of the fracture geometry can be challenging with other methods20, but using a saw allows for more control for the surgeon and alleviates the variability in ensuing fractures. The saw approach has also been used to create metaphyseal fractures of the proximal mouse femurs successfully21.
Differences between male and female mice in their response to fracture healing are rarely observed. However, sex may become a factor with age and drug intervention, so comparing animals of the same sex or performing statistics to interrogate sex differences before combining samples is strongly recommended. Furthermore, this protocol was designed for C57BL6/J mice. Investigators using other mouse strains should compare healing in male and female mice to identify any sex differences.
We believe this femur fracture surgery is an efficient model to recapitulate the significant healing steps in mice and can be used to test the effects of genetic modifications or therapeutic interventions on fracture recovery in humans.

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><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 &amp;frac14; (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 &amp;micro;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 (Figure 3). At 5 days post fracture (dpf), Alcian Blue staining can be used to visualize the soft callus and subsequently quantify the cartilage area&#160;(Figure 2A,B). Mineralization is detectable by microCT at 28 dpf (Figure 2C). The volume of the mineralized callus, the distance of the fracture gap, and the bone strength measured by mechanical testing are commonly used as quantifiable outcomes of fracture repair. Genetic modification or drug intervention can change the course of recovery, so performing a time-course study to characterize fractures at different stages of repair is recommended. The whole callus can be dissected for molecular analysis and the contralateral bone shaft can be used as a control. &#160;If the fracture ends are not aligned or adequately secured with the pin, the resulting images will show a lack of callus formation on all or one side of the fracture site&#160;(Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63074/63074fig01.jpg" /> 
Figure 1: Fracture and insertion of the stabilizing pin. (A) A square is shaved on the right leg of a C57BL/6J mouse. (B) After an incision is made in the skin and fascia, curved forceps are secured underneath the femur to separate the muscle, skin, and bone. (C) After the cut is made, two fracture ends are created: the proximal section of the femur attached to the hip bone and the distal section attached to the knee. The guide needle (green) is inserted into the distal section and pushed through the knee joint. (D) The guide needle is removed from the distal section, inserted in the proximal section, and pushed through the hip joint. (E) The stabilizing pin (grey needle) is inserted into the guide needle protruding from the hip joint. (F) The stabilizing pin is pushed through the proximal section, into the distal section, and through the knee joint using the path made by the guide needle in C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63074/63074fig01large.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/63074/63074fig02.jpg" /> 
Figure 2: Histology and microCT of femur fracture. (A) Formalin-fixed paraffin sections of femur fractures were collected at 5, 10, and 28 dpf and stained with Alcian Blue/Eosin/Orange G. Scale bar = 500 &#956;m. (B) Cartilage area was quantified using ImageJ software at 5, 10, and 28 dpf. (C) At 28 dpf, mineralization was observed, and the callus volume and fracture gap could be measured by microCT. Scale bar = 1,000 &#956;m. Data shown as Mean &#177; SEM. The mineralized callus volume was measured by contouring around the cortical bone at the fracture site. The dark grey area delineates the mineralized callus on the image, while the cortical bone (light grey) is not included in the measurement. Data shown as Mean&#160;&#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63074/63074fig02large.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/63074/63074fig03.jpg" /> 
Figure 3: Fluorescent reporter model used to visualize expansion of Prx1+ periosteal cells after fracture. Prx1CreER; Rosa26tdTomato mice were injected daily for five days with 80 mg/kg body weight of tamoxifen to induce tdTomato expression. Three days after the final injection, femur fracture was initiated, and mice were sacrificed at 7 or 14 dpf to track where Prx1-expressing cells and their progeny (Prx1+) are located within the fracture callus and expanded periosteum. Scale bar = 500 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63074/63074fig03large.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/63074/63074fig04.jpg" /> 
Figure 4: Example of irregular healing due to surgical issues. Fracture ends were not aligned properly and the stabilizing pin pierced through the proximal section of the femur in this example. These errors resulted in callus formation where the femur was pierced (yellow box)&#160;rather than at the cut site. Scale bar = 500 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63074/63074fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Composition of the solutions required for histology. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63074/63074_Supplemetary File 1.docx" target="_blank">Please click here to download this File.</a>

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 ...

transverse-fracture-of-the-mouse-femur-with-stabilizing-pin

Nanyang Technological University

Research

JoVE Journal

Biology

3.4K Views.  Harvard School of Dental Medicine. Dit protocol beschrijft een betrouwbare techniek om femurfracturen bij volwassen muizen uit te voeren en te bestuderen. Deze techniek is eenvoudig, snel en vereist minimale gereedschappen om te worden uitgevoerd. Het maakt de karakterisering van verschillende stadia van fractuurgenezing mogelijk.Individuen kunnen in eerste instantie worstelen met het goed uitlijnen van de fractuuruiteinden en het creëren van consistente fracturen tussen muizen. Door goed te letten op de plaats van de ...

Video: Transverse Fracture of the Mouse Femur with Stabilizing Pin

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

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 ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

Bioengineering

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 ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

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 ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

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 ...

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

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