The research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number F30AR071201 and R01AR066028.

This protocol was developed to ensure that animals are not used needlessly and are spared all unnecessary pain and distress; it adheres to all applicable federal, state, local, and institutional laws and guidelines governing animal research. The protocol was developed under the guidance of a university-wide Laboratory Animal Medicine Program directed by veterinarians specialized in laboratory animal medicine. The protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
1. Fracture Tower Construction
Note: All parts are listed in the materials section (Table of Materials). Detailed technical drawings are provided for the machined and 3D-printed parts in Supplementary Figures 1-12. The subassembly technical drawings include fastener details for all mounted parts (Supplementary Figures 1, 2, 7, and 9).
<ol>
	<li>Support subassembly 
	NOTE: For a technical drawing of the support subassembly, see Supplementary Figure 1.
	<ol>
		<li>Attach the Beam Support--Jaw Section at the midpoint of the Beam Support--Horizontal Section.</li>
		<li>Attach the Beam Support--Vertical 1 to the top surface of the Beam Support--Jaw Section, 2 in from the Beam Support--Horizontal Section.</li>
		<li>Attach the Beam Support--Vertical 2 to the top surface of the Beam Support--Horizontal Section at the midpoint (7 in from the end).</li>
		<li>Attach the Beam, Support--Plate Mount to the end of both Beam Support--Vertical 1 and Beam Support--Vertical 2. The end of the plate support should be flush with the back of Beam Support--Vertical 2.</li>
	</ol>
	</li>
	<li>Ram subassembly 
	Note: For a technical drawing of the ram subassembly, see Supplementary Figure 2.
	<ol>
		<li>Machine the Block Stop and the Block Guide (Supplementary Figure 3); the Rod Ram (Supplementary Figure 4); the Screw Alignment (Supplementary Figure 5); and the Plate Mounting (Supplementary Figure 6).</li>
		<li>Attach the Plate Mounting to the Beam Support--Plate Mount of the support subassembly.</li>
		<li>In the following order, slide the first Linear Sleeve Bearing; the Block Guide; the second Linear Sleeve Bearing; and the Block Stop on the Rod Ram. Attach the guides and the blocks to the Plate Mounting.</li>
		<li>Attach three &#8540;-in nuts to the threaded portion of the Rod Ram. One should be flush with the end of the rod to engage with the electromagnet. The other 2 will be used to adjust the fracture depth.</li>
		<li>Align the grove in the Rod Ram to face forward and insert the Alignment Screw into the threaded hole of the Block Guide.</li>
	</ol>
	</li>
	<li>Magnet subassembly 
	Note: For a technical drawing of the magnet subassembly, see Supplementary Figure 7.
	<ol>
		<li>Solder the Electromagnet leads to the wire (polarity is not a factor for the electromagnet operation). Allow enough length to reach the floor, where the fracture device will be positioned. Use zip ties or another form of attachment to stress relieve the wire.</li>
		<li>Strip the Power Supply&#39;s end and connect it to the Foot Pedal. Finally, connect the wire to the Foot Pedal in an &#34;off&#34; (normally open) configuration. Test the circuit to ensure the Electromagnet is on when the Foot Switch is not pressed. This will hold the ram up before the fracture.</li>
		<li>Print the Mount Magnet (Supplementary Figures 8A and 8B) using an additive manufacturing device, or machine the part from aluminum.</li>
		<li>Attach the Electromagnet to the Mount Magnet.</li>
		<li>Attach 2 Corner Brackets to the Beam Support--Magnet.</li>
		<li>In the following order, thread the Rod Magnet through the top Corner Bracket and add one &#188;-in nut; the Mount Magnet; two &#188;-in nuts; and the bottom Corner Bracket. Secure the assembly with two &#188;-in nuts on each end.</li>
	</ol>
	</li>
	<li>Complete assembly 
	Note: For a technical drawing of the complete assembly, see Supplementary Figure 9.
	<ol>
		<li>Attach the Magnet Subassembly to the top surface of the Beam, Support--Plate Mount.</li>
		<li>Adjust the alignment of the Beam Support--Magnet so the magnet engages with the Rod, Ram. 
		NOTE: If the rod does not release when the foot pedal is pressed, reduce the contact area between the electromagnet and the rod by moving the Beam Support--Magnet.</li>
		<li>Machine the Brackets Leg Jaw (Supplementary Figure 10).</li>
		<li>Attach the two Brackets Leg Jaw to the Beam Support--Jaw Section. When dropped, the tip of the ram should be at an equal distance from each jaw.</li>
		<li>Place the Platform Fracture (Supplementary Figures 11A and 11B) above the jaws.</li>
		<li>Print the Jig Positioning Fracture (Supplementary Figures 12A and 12B) and the Jig Pin Gauge (Supplementary Figures 13A and 13B) using an additive manufacturing device, or machine the parts from aluminum. 
		NOTE: The dimensions of the Jigs will be calculated in the optimization steps detailed in step 2.</li>
		<li>Attach the Jig Positioning Fracture to the Platform Fracture.</li>
		<li>Confirm that the depth of the impact can be adjusted using the two stop nuts on the Rod Ram.</li>
		<li>Confirm that the speed of the impact can be adjusted by moving the Mount Magnet up and down.</li>
		<li>Confirm that the width of the fracture can be adjusted by moving the Brackets Leg Jaw closer or further away from the Rod Ram.</li>
	</ol>
	</li>
</ol>
2. Fracture Optimization
<ol>
	<li>Fracture location
	<ol>
		<li>Obtain radiographs of the limb (femur or tibia) to be fractured in a representative sample of 5 euthanized animals. 
		NOTE: The sample should be matched to the specimens, which will be used in the experimental protocol based on age, genotype, and sex. Even if the final protocol calls for only one fractured limb, both sample limbs will be used.</li>
		<li>Position the limb tangential to the x-ray beam to acquire true-lateral and anterior/posterior views to the bone. Place an object of known dimension at the imaging plane to provide a scale for analysis.</li>
		<li>Note: If imaging femurs, ensure the limb is in full extension, where the femur is in the same axial plane as the tibia.</li>
		<li>Mark the desired location of the fracture on the radiograph of the limb to be fractured (Figure 1A - dashed line). Measure from the calcaneal-tibial joint to the level of the marked fracture (Figure 1A). Calculate the mean fracture length (FL) for all trial specimens. Measure from the intercondylar notch for femur fractures.</li>
	</ol>
	</li>
	<li>Fracture-positioning jig
	<ol>
		<li>Measure the distance from the outside surface of one support anvil to the center of the guillotine impact (CGI) (Figure 2). Subtract the CGI from the FL, described in step 2.1.4, to calculate the fracture-positioning jig depth (JD). Machine or 3D-print a U-shaped channel with a height and a width equal to the anvil, and a depth equal to the JD (Figure 3A). A sample technical drawing and CAD file are included in Supplementary Figures 12A and 12B. 
		NOTE: When the limb is placed in the jig, the dorsum of the foot should lie against the surface furthest from the guillotine impact. Modify the U-shaped channel if additional clearance is required for the limb.</li>
		<li>Position the specimen in the fracture apparatus in the prone position for femur fractures or in the supine position for tibia fractures (Figure 4). Press the dorsum of the foot against the end of the fracture-positioning jig. Manually depress the guillotine until the limb fractures. Obtain a radiograph of the fractured limb to confirm the jig size and fracture location (Figure 2B).</li>
		<li>Increase JD if the fracture location is too distal on the bone, or decrease JD if the fracture location is too proximal on the bone.</li>
	</ol>
	</li>
	<li>Stabilization of the pin parameters
	<ol>
		<li>Pin length:&#160;Using the radiographs obtained in step 2.1, measure the limb length (LL) from the tibial plateau to the level of the posterior malleolus for tibia fractures, or the intercondylar notch to the greater trochanter for femur fractures. Multiply the bone length by 0.9 to calculate the pin length (PL) (Figures 1A and 3B).</li>
		<li>Pin Width: Using the radiographs obtained in step 2.1, measure the minimum medullary diameter (MD) in the fractured limb (Figure 1A). Select a needle with a gauge approximately equivalent to the medullary diameter and a length more than 1.5 x PL. 
		NOTE: An approximate pin size for a 14-week-old C57BL/6J mouse is 22 G, 1&#189; in and 27 G, 1&#188; in for femur and tibia, respectively.</li>
	</ol>
	</li>
	<li>Pin cutting gauge
	<ol>
		<li>2.4.1. Machine or 3D-print a gauge with a length equal to PL minus the needle length (CGL) (Figure 3B; Supplementary Figures 13A and 13B). One end should have an overhang to rest against the hub of the needle and the other should indicate where the pin should be cut. A sample technical drawing and CAD file are included in Supplementary Figures 13A and 13B.</li>
	</ol>
	</li>
	<li>Intramedullary pin fracture stabilization
	<ol>
		<li>Using the non-fractured trial specimens from step 2.1, remove hair with an electric clipper or depilatory cream from mid-tibia to mid-femur, exposing the knee joint.</li>
		<li>Tibia pinning: Insert the needle percutaneously lateral to the patellar ligament. Retract the patellar ligament medially and align the tip of the needle to the axis of the tibia. Using a reaming motion, gently breach the tibial plateau and guide the needle down the medullary cavity.</li>
		<li>Femur pinning: Insert the needle percutaneously lateral to the patellar ligament. Retract the patellar ligament medially and align the tip of the needle to the axis of the femur in the intercondylar notch. Using a reaming motion, gently breach the articular surface of the intercondylar notch and guide the needle down the medullary cavity.</li>
		<li>Using the gauge manufactured in step 2.4, ream until the exposed needle is equal to the gauge length. Retract the needle to provide enough room (~3 mm) to cut the needle at the level indicated by the gauge. 
		NOTE: Be sure to hold the proximal (plastic) end of the needle while cutting, so it does not become a hazardous projectile.</li>
		<li>Crimp 0.3 mm of the distal end of the pin using a pin cutter and then cut the pin at the level of the gauge.&#160;Sink the pin to the articular surface using a rod with a diameter 1.5x larger than the diameter of the needle. 
		NOTE: Crimping prevents rotation of the needle and migration by increasing the needle-bone contact.</li>
		<li>Obtain radiographs to confirm the needle extends the length of the medullary canal of the limb and does not protrude from the proximal or distal end (Figure 1C).</li>
	</ol>
	</li>
	<li>Impact depth
	<ol>
		<li>Using the radiographs obtained in step 2.1, measure the diameter of the cortex at the level of the desired fracture (Figure 1A). Calculate the mean cortical diameter (CD) for all trial specimens.</li>
		<li>Position a pinned trial specimen from step 2.5 in the fracture device with the fracture-positioning jig manufactured in step 2.2. Rest the impact ram on the uninjured limb. 
		NOTE: Do not allow the ram to drop; the bone should remain intact during this optimization step.</li>
		<li>Apply enough downward force on the ram to compress soft tissue, but not fracture the bone. Adjust the impact depth (ID) to 0.75 x CD (Figure 2). 
		NOTE: The ideal impact depth is 0.5 x CD when fracturing a bone without any soft tissue. Using 0.75 accounts for the additional soft-tissue compression.</li>
	</ol>
	</li>
	<li>Anvil width
	<ol>
		<li>Set the anvil width (AW) to 0.4 cm for the mouse tibia or femur (Figure 2). 
		NOTE: A wider width is recommended for larger specimens such as rats.</li>
	</ol>
	</li>
	<li>Ram weight
	<ol>
		<li>A minimum weight of 250 g is recommended for murine specimens. 
		NOTE: Additional weight can be threaded on to the ram for larger specimens (Figure 2).</li>
	</ol>
	</li>
	<li>Impact velocity
	<ol>
		<li>Set the drop height (DH) to 2 cm (Figure 2). Position the ram in its starting position by connecting it to the activated electromagnet.</li>
		<li>Position a trial limb in the fracture apparatus. Press the dorsum of the foot against the fracture-positioning jig manufactured in step 2.2. Briefly depress the footswitch to release the ram and then reset it to its starting position.</li>
		<li>Radiograph the impacted trial limb. Analyze the limb for any evidence of a fracture (Figure 1D). 
		NOTE: This can be subtle when using low velocities with a controlled impact depth.</li>
		<li>If no fracture is generated, repeat steps 2.9.1 - 2.9.3 and increase the drop height by 2 cm.</li>
		<li>If a fracture is generated, record the drop height, and multiply it by 1.1. This is the new DH.</li>
		<li>Using the DH from step 2.9.5, fracture the next trial limb.</li>
		<li>If no fracture is generated, repeat steps 2.9.1 - 2.9.6 and increase the drop height by 2 cm.</li>
		<li>If a fracture is generated, repeat steps 2.9.6 - 2.9.7 until all test samples are used. Record the final DH and all parameters (FL, CGI, JD, PL, MD, PS, CGL, CD, ID, AW, and RW) from the optimization. Record the trial specimens&#39; age, sex, genotype, and weight.</li>
	</ol>
	</li>
</ol>
3. Closed-stabilized Fracture Generation
<ol>
	<li>Set-up
	<ol>
		<li>Sterilize all equipment and instruments via autoclave, hot bead immersion, or their equivalent.</li>
		<li>Place a heating element on the surgical table and set it to the optimal temperature. Cover the element with a surgical drape. Prepare 3 x 3 in2 of surgical drape with a 0.75-in circle cut out in the middle.</li>
		<li>Confirm the adjustment of the fracture tower before each trial (Figure 2). Set the ID, AW, RW, and DH to the values derived from the optimization protocol specific to the sex, age, and genotype for the specimen to be studied.</li>
		<li>Weigh and record the weight of the animal.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Adequately sedate the mouse using inhalant anesthetics (isoflurane: 4 - 5% for induction; 1 - 2% for maintenance) or another established laboratory anesthesia protocol. The respiratory rate should be 55 - 100 breaths/min. The animal should not be responsive to a hind-limb toe pinch.</li>
		<li>Administer the first dose of the postoperative analgesia buprenorphine (0.1 mg/kg subcutaneously).</li>
		<li>Apply ocular lubrication to prevent corneal drying.</li>
		<li>Remove the animal&#39;s hair with an electric clipper from mid-tibia to mid-femur, exposing the knee joint. Clean the site of excess hair using non-reactive tape. Prepare the pinning site with a wet swab moistened with 70% EtOH. Repeat as necessary to remove all hair from the incision area.</li>
		<li>Prepare and clean the pinning area with alternative swabs of povidone-iodine and 70% EtOH. Use two alternative swab sequences to ensure sterility.</li>
		<li>A drape is then placed around the surgical site after the skin has been appropriately disinfected.</li>
		<li>Pin the limb to be fractured using the protocol described in step 2.5. Acquire radiographs to confirm the pin extends the length of the medullary canal but does not protrude from the proximal or distal end.</li>
		<li>Turn on the electromagnet and connect the impact ram to place it in the starting position.</li>
		<li>Position the specimen in the fracture apparatus by placing it in a prone position for femur fractures or in a supine position for tibia fractures. The pinned limb should be placed across the anvils and in the fracture-positioning jig with the dorsum of the foot pressed against the outside of the jig.</li>
		<li>While pressing the foot with one hand and ensuring only the limb is in the impact ram target area, briefly depress the footswitch to release the ram. Replace the ram in the starting position.</li>
		<li>Acquire radiographs and confirm the fracture location and type.</li>
	</ol>
	</li>
	<li>Postoperative management
	<ol>
		<li>Monitor the animal every 15 min during its recovery from anesthesia until the animal is conscious, can maintain sternal recumbency, and is ambulatory. Confirm the animal is able to ambulate over a 72-h period.</li>
		<li>House the animal individually until it has completely recovered.</li>
		<li>Maintain analgesia over a 48-h period with buprenorphine (0.1 mg/kg subcutaneously) administered every 12 h.</li>
		<li>Monitor and record the health status of the animal daily for 7 - 10 d or until euthanasia.</li>
	</ol>
	</li>
	<li>Post-fracture analysis
	<ol>
		<li>Measure FL, PL, CD, MD, and the fracture pattern. Record the measurements in a master data file.</li>
	</ol>
	</li>
</ol>

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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=Fractures+in+geriatric+mice+show+decreased+callus+expansion+and+bone+volume.">Fractures in geriatric mice show decreased callus expansion and bone volume.</a> Clinical Orthopaedics and Related Research. 472 (11), 3523-3532 (2014).</li><li>Bonnarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+closed+fracture+in+laboratory+animal+bone.">Production of a standard closed fracture in laboratory animal bone.</a> Journal of orthopaedic research. 2 (1), 97-101 (1984).</li><li>Marturano, J. 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Journal Canadien De Chirurgie. 13 (4), 415-420 (1970).</li><li>Byrne, M., Cleveland, B., Marturano, J., Wixted, J., Billiar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+reproducible+murine+femoral+fracture+device.+Conference:+Bioengineering+Conference,+2007.">Design of a reproducible murine femoral fracture device. Conference: Bioengineering Conference, 2007.</a> NEBC '07. IEEE 33rd Annual Northeast. , (2007).</li><li>Carter, D. R., Hayes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compact+bone+fatigue+damage-I.+Residual+strength+and+stiffness.">Compact bone fatigue damage-I. Residual strength and stiffness.</a> Journal of Biomechanics. 10 (5), 325-337 (1977).</li><li>McGee, A., Qureshi, A., Porter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+biomechanics+and+patterns+of+limb+fractures.">Review of the biomechanics and patterns of limb fractures.</a> Trauma. 6 (1), 29-40 (2004).</li></ol>

The authors have nothing to disclose.

This fracture optimization and generation protocol provides researchers with an efficient method to derive at fracture parameters and perform a minimally invasive procedure, which produces precise, repeatable, transverse fractures. Additionally, this protocol establishes a common set of fracture generation parameters, which promotes method consistency amongst researchers. These parameters will enable the creation of a common fracture database to establish fracture standards based on a variety of parameters (e.g....

Research on fracture healing is necessary to address a large clinical and economic problem. Each year over 12 million fractures are treated in the United States1, costing $80 billion per year2. The likelihood of a male or female suffering a fracture in their lifetime is 25% and 44%, respectively3. Problems associated with fracture healing are expected to increase with increased comorbidities as the population ages. To study and address this problem, robust models of fracture generation and stabilization are required. Rodent models are ideally suited for this purpose. They provide clinical relevance and can be modified to address specific conditions (i.e., multiple injuries, open, closed, ischemic, and infected fractures). In addition to replicating clinical scenarios, animal fracture models are important for understanding bone biology and developing therapeutics and devices. However, attempts to study differences between interventions may be complicated by the variability introduced by inconsistent fracture generation. Thus, generating reproducible and consistently closed fractures in animal models is essential to the field of musculoskeletal research.
Despite properly controlling for potential subject heterogeneity by ensuring the appropriate genetic background, sex, age, and environmental conditions, the production of clinically-relevant consistent bone injuries is a significant variable affecting reproducibility that must be controlled. Statistical comparisons using inconsistent fractures are plagued with experimental noise and a high variability4; in addition, fracture variability can result in unnecessary animal death because of the need to increase the sample size or the necessity to euthanize animals with comminuted or malpositioned fractures. The purpose of the method described herein is to optimize the fracture generation parameters that are specific to sample type and yield a consistent fracture location and pattern.
Current models of fracture generation fall into two broad categories, each with their own strengths and weaknesses. Open-fracture (osteotomy) models undergo surgery to expose the bone, after which a fracture is induced by cutting the bone or weakening it and then manually breaking it5,6,7,8. The benefits of this method are the direct visualization of the fracture site and a more consistent fracture location and pattern. However, the physiological and clinical relevance of the approach and mechanism of injury is limited. Additionally, open methods of fracture generation require a surgical approach and closure with prolonged periods during which the rodents are exposed to an increased risk of contamination.
Closed techniques address many of the open technique&#39;s limitations. Closed techniques produce fractures using an externally applied blunt force trauma which induces injury to the bone and surrounding tissues, more similar to those seen in human clinical injuries. The most common method was described by Bonnarens and Einhorn in 19849. They described a weighted guillotine being used to impart blunt trauma to break the bone without causing any external skin wounds. This method has been widely adopted to study the effect of genetics10,11, pharmacologic therapy12,13,14,15, mechanics16,17, and physiology18,19,20 on bone healing in mice and rats. While the benefit of closed methods is physiologically relevant fractures, experimental reproducibility and rigor are limited by fracture heterogeneity. The inconsistent fracture generation results in a limited between-group differentiation, lost specimens, and an increase in animals needed to achieve statistical significance.
Controlling the variability in fracture generation and stabilization is essential to produce meaningful results. In order to properly study the biology of fracture repair, a simple yet robust fracture model is needed. The model should be translatable to rodent species, bone types (femur or tibiae, for example), and across variable mouse genetic backgrounds and induced mutations. Furthermore, the ideal procedure should be technically simple and produce consistent results. To address fracture heterogeneity, the method described herein is the construction of a well-controlled fracture device that can then be used to optimize parameters and generate consistently closed fractures regardless of age, sex, or genotype.

<table><tbody><tr><td>&lt;strong&gt;Support Subassembly&lt;/strong&gt;</td><td></td><td></td><td>Supplementary Figure 1</td></tr><tr><td>Beam, Support--Jaw Section&amp;nbsp;</td><td>80/20</td><td>1003 x 9.00</td><td>w/ #7042 at A, C, in Left End</td></tr><tr><td>Beam, Support--Horizontal Section</td><td>80/20</td><td>1002 x 14.00</td><td></td></tr><tr><td>Beam, Support--Vertical 1</td><td>80/20</td><td>1050 x 10.50&amp;nbsp;</td><td>w/ #7042 at A in Left End and at A in Right End</td></tr><tr><td>Beam, Support--Vertical 2</td><td>80/20</td><td>1010 x 10.50&amp;nbsp;</td><td>w/ #7042 at D, B in Left End and at A in Right End</td></tr><tr><td>Beam, Support--Plate Mount</td><td>80/20</td><td>1030 x 8.00&amp;nbsp;</td><td>w/ #7036 at Left End</td></tr><tr><td>Beam, Support--Magnet</td><td>80/20</td><td>1010 x 13.50&amp;nbsp;</td><td>w/ #7042 at A, C, in Right End</td></tr><tr><td>Anchors (3)</td><td>80/20</td><td>3392</td><td></td></tr><tr><td>Double Anchor (3)</td><td>80/20</td><td>3091</td><td></td></tr><tr><td>Bolt Assembly (6)</td><td>80/20</td><td>3386</td><td>1/4-20 x 3/8&quot;</td></tr><tr><td>Button Head Socket Cap Screw (6)</td><td>80/20</td><td>3604</td><td>1/4-20 x 3/4&quot;</td></tr><tr><td>&lt;strong&gt;Ram Subassembly&lt;/strong&gt;</td><td></td><td></td><td>Supplementary Figure 2</td></tr><tr><td>Block, Stop</td><td>Custom</td><td></td><td>Supplementary Figure 3</td></tr><tr><td>Block, Guide</td><td>Custom</td><td></td><td>Supplementary Figure 3</td></tr><tr><td>Rod, Ram</td><td>Custom</td><td></td><td>Supplementary Figure 4</td></tr><tr><td>Alignment Screw</td><td>Custom</td><td></td><td>Supplementary Figure 5</td></tr><tr><td>Plate, Mounting</td><td>Custom</td><td></td><td>Supplementary Figure 6</td></tr><tr><td>Linear Sleeve Bearing (2)</td><td>McMaster-Carr</td><td>8649T2</td><td></td></tr><tr><td>Hex Nut (3)</td><td>McMaster-Carr</td><td>92673A125</td><td>3/8-16 UNC</td></tr><tr><td>Socket Cap Screw (8)</td><td>McMaster-Carr</td><td>92196A108</td><td>4/40 x 3/8&quot;</td></tr><tr><td>Socket Cap Screw (6)</td><td>McMaster-Carr</td><td>92196A032</td><td>4/40 x 1 1/8&quot;</td></tr><tr><td>Socket Cap Screw (1)</td><td>McMaster-Carr</td><td>92196A267&amp;nbsp;</td><td>10/32 3/8&quot;</td></tr><tr><td>&lt;strong&gt;Magnet Subassembly&lt;/strong&gt;</td><td></td><td></td><td>Supplementary Figure 7</td></tr><tr><td>Mount, Magnet</td><td>Custom</td><td></td><td>Supplementary Figure 8</td></tr><tr><td>Power Supply</td><td>McMaster-Carr</td><td>70235K23</td><td></td></tr><tr><td>Foot Switch</td><td>McMaster-Carr</td><td>7376k2</td><td></td></tr><tr><td>Electromagnet</td><td>McMaster-Carr</td><td>5698k111</td><td></td></tr><tr><td>Wire - 10 feet</td><td>McMaster-Carr</td><td>9936k12</td><td></td></tr><tr><td>Rod, Magnet</td><td>McMaster-Carr</td><td>95412A566</td><td>1/4&quot; Threaded Rod x 7&quot;</td></tr><tr><td>Corner Bracket (6)</td><td>80/20</td><td>4108</td><td></td></tr><tr><td>Socket Cap Screw (1)</td><td>McMaster-Carr</td><td>92196A705</td><td>10/32 1 1/4&quot;</td></tr><tr><td>Hex Nut (4)</td><td>McMaster-Carr</td><td>92673A113</td><td>1/4-20 UNC</td></tr><tr><td>&lt;strong&gt;Complete Assembly&lt;/strong&gt;</td><td></td><td></td><td>Supplementary Figure 9</td></tr><tr><td>Bracket, Leg Jaw (2)</td><td>Custom</td><td></td><td>Supplementary Figure 10</td></tr><tr><td>Platform, Fracture</td><td>Custom</td><td></td><td>Supplementary Figure 11</td></tr><tr><td>Jig, Positioning-Fracture</td><td>Custom</td><td></td><td>Supplementary Figure 12</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pin Cutter</td><td>Medical Supplies and Equipment</td><td>150S</td><td></td></tr><tr><td>Needles</td><td>Sigma</td><td>Z192430, Z192376&amp;nbsp;</td><td>23g x 1.5&quot; - mouse femur, 27g x 1.25&quot; - mouse tibia</td></tr></tbody></table>

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 guillotine previously used in our laboratory was developed in 2004 and was based on models published by Einhorn21. The design did not permit adjustments to adequately account for any differences in bone morphology and did not permit a reproducible positioning of the limb. Furthermore, the previous apparatus required two people to operate it. Therefore, we designed, engineered, and built a new fracture apparatus. The main design goal was the possibility to the h...

Watch this Scientific Journal Video about Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents at JoVE.com

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

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 ...

fracture-apparatus-design-protocol-optimization-for-closed-stabilized

Research

JoVE Journal

Medicine

13.3K Views.  Michigan State University. 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.

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

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

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 ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Bioengineering

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. ...

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 ...

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 ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

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 ...

Biology

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 ...