Name: fracture apparatus design and protocol optimization for closed-stabilized fractures in rodents
Uploaded: 2018-08-14T15:00:00
Description: Watch this Scientific Journal Video about Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents at JoVE.com

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).
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<ol>
	<li>Corso, P., Finkelstein, E., Miller, T., Fiebelkorn, I., Zaloshnja, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+lifetime+costs+of+injuries+in+the+United+States.">Incidence and lifetime costs of injuries in the United States.</a> Injury Prevention. 12 (4), 212-218 (2006).</li><li>Nguyen, N. D., Ahlborg, H. G., Center, J. R., Eisman, J. A., Nguyen, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+lifetime+risk+of+fractures+in+women+and+men.">Residual lifetime risk of fractures in women and men.</a> Journal of Bone and Mineral Research: The Official Journal of the American Society for Bone and Mineral Research. 22 (6), 781-788 (2007).</li><li>Thompson, Z., Miclau, T., Hu, D., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+intramembranous+ossification+during+fracture+healing.">A model for intramembranous ossification during fracture healing.</a> Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 20 (5), 1091-1098 (2002).</li><li>Cheung, K. M. C., Kaluarachi, K., Andrew, G., Lu, W., Chan, D., Cheah, K. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> Journal of Orthopaedic Research. 21 (4), 685-690 (2003).</li><li>Connolly, C. K., 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=A+reliable+externally+fixated+murine+femoral+fracture+model+that+accounts+for+variation+in+movement+between+animals.">A reliable externally fixated murine femoral fracture model that accounts for variation in movement between animals.</a> Journal of Orthopaedic Research. 21 (5), 843-849 (2003).</li><li>Histing, T., 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=An+internal+locking+plate+to+study+intramembranous+bone+healing+in+a+mouse+femur+fracture+model.">An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.</a> Journal of Orthopaedic Research. 28 (3), 397-402 (2010).</li><li>Gr&#246;ngr&#246;ft, I., 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=Fixation+compliance+in+a+mouse+osteotomy+model+induces+two+different+processes+of+bone+healing+but+does+not+lead+to+delayed+union.">Fixation compliance in a mouse osteotomy model induces two different processes of bone healing but does not lead to delayed union.</a> Journal of Biomechanics. 42 (13), 2089-2096 (2009).</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>Huang, C., 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=The+spatiotemporal+role+of+COX-2+in+osteogenic+and+chondrogenic+differentiation+of+periosteum-derived+mesenchymal+progenitors+in+fracture+repair.">The spatiotemporal role of COX-2 in osteogenic and chondrogenic differentiation of periosteum-derived mesenchymal progenitors in fracture repair.</a> PloS One. 9 (7), 100079 (2014).</li><li>Waki, T., 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=Profiling+microRNA+expression+during+fracture+healing.">Profiling microRNA expression during fracture healing.</a> BMC Musculoskeletal Disorders. 17, 83 (2016).</li><li>Yee, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sclerostin+antibody+treatment+improves+fracture+outcomes+in+a+Type+I+diabetic+mouse.">Sclerostin antibody treatment improves fracture outcomes in a Type I diabetic mouse.</a> Bone. 82, 122-134 (2016).</li><li>Wong, E., 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=A+novel+low-molecular-weight+compound+enhances+ectopic+bone+formation+and+fracture+repair.">A novel low-molecular-weight compound enhances ectopic bone formation and fracture repair.</a> The Journal of Bone and Joint Surgery. American Volume. 95 (5), 454-461 (2013).</li><li>Prodinger, P. M., 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=Does+Anticoagulant+Medication+Alter+Fracture-Healing?+A+Morphological+and+Biomechanical+Evaluation+of+the+Possible+Effects+of+Rivaroxaban+and+Enoxaparin+Using+a+Rat+Closed+Fracture+Model.">Does Anticoagulant Medication Alter Fracture-Healing? A Morphological and Biomechanical Evaluation of the Possible Effects of Rivaroxaban and Enoxaparin Using a Rat Closed Fracture Model.</a> PloS One. 11 (7), 0159669 (2016).</li><li>Menzdorf, L., 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=Local+pamidronate+influences+fracture+healing+in+a+rodent+femur+fracture+model:+an+experimental+study.">Local pamidronate influences fracture healing in a rodent femur fracture model: an experimental study.</a> BMC Musculoskeletal Disorders. 17, 255 (2016).</li><li>Hagiwara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+stability+dictates+the+differentiation+pathway+of+periosteal+progenitor+cells+in+fracture+repair.">Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.</a> Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 33 (7), 948-956 (2015).</li><li>Gardner, M. J., 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=Differential+fracture+healing+resulting+from+fixation+stiffness+variability:+a+mouse+model.">Differential fracture healing resulting from fixation stiffness variability: a mouse model.</a> Journal of Orthopaedic Science: Official Journal of the Japanese Orthopaedic Association. 16 (3), 298-303 (2011).</li><li>Catma, M. F., 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=Remote+ischemic+preconditioning+enhances+fracture+healing.">Remote ischemic preconditioning enhances fracture healing.</a> Journal of Orthopaedics. 12 (4), 168-173 (2015).</li><li>Lichte, P., 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=Impaired+Fracture+Healing+after+Hemorrhagic+Shock.">Impaired Fracture Healing after Hemorrhagic Shock.</a> Mediators of Inflammation. 2015, 132451 (2015).</li><li>Lopas, L. 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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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.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Support Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td style="width:315px;">Supplementary Figure 1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Jaw Section&#160;</td>
			<td style="width:209px;">80/20</td>
			<td>1003 x 9.00</td>
			<td>w/ #7042 at A, C, in Left End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Horizontal Section</td>
			<td style="width:209px;">80/20</td>
			<td>1002 x 14.00</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Vertical 1</td>
			<td style="width:209px;">80/20</td>
			<td>1050 x 10.50&#160;</td>
			<td>w/ #7042 at A in Left End and at A in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Vertical 2</td>
			<td style="width:209px;">80/20</td>
			<td>1010 x 10.50&#160;</td>
			<td>w/ #7042 at D, B in Left End and at A in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Plate Mount</td>
			<td style="width:209px;">80/20</td>
			<td>1030 x 8.00&#160;</td>
			<td>w/ #7036 at Left End</td>
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		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Magnet</td>
			<td style="width:209px;">80/20</td>
			<td>1010 x 13.50&#160;</td>
			<td>w/ #7042 at A, C, in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Anchors (3)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3392</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Double Anchor (3)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3091</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Bolt Assembly (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3386</td>
			<td>1/4-20 x 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Button Head Socket Cap Screw (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3604</td>
			<td>1/4-20 x 3/4&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ram Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 2</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Block, Stop</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 3</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Block, Guide</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 3</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Rod, Ram</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 4</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Alignment Screw</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 5</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Plate, Mounting</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 6</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Linear Sleeve Bearing (2)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">8649T2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Hex Nut (3)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>92673A125</td>
			<td>3/8-16 UNC</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (8)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A108</td>
			<td>4/40 x 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (6)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A032</td>
			<td>4/40 x 1 1/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (1)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A267&#160;</td>
			<td>10/32 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Magnet Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 7</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Mount, Magnet</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 8</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Power Supply</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>70235K23</td>
			<td></td>
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		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Foot Switch</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>7376k2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Electromagnet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>5698k111</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Wire - 10 feet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">9936k12</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rod, Magnet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>95412A566</td>
			<td>1/4&#34; Threaded Rod x 7&#34;</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Corner Bracket (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">4108</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (1)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A705</td>
			<td>10/32 1 1/4&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Hex Nut (4)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>92673A113</td>
			<td>1/4-20 UNC</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Complete Assembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 9</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Bracket, Leg Jaw (2)</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 10</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Platform, Fracture</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 11</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Jig, Positioning-Fracture</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 12</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Other</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Pin Cutter</td>
			<td style="width:209px;">Medical Supplies and Equipment</td>
			<td style="width:147px;">150S</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Needles</td>
			<td style="width:209px;">Sigma</td>
			<td style="width:147px;">Z192430, Z192376&#160;</td>
			<td>23g x 1.5&#34; - mouse femur, 27g x 1.25&#34; - 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 ...

This method can help answer key questions in the field of bone biology, such as which interventions promote fracture healing. The main advantage of this technique is that it provides a simple method to derive parameters to generate consistent fractures. To locate the region for the fracture, obtain radiographs of the limb femur or tibia to be fractured in a representative sample of five euthanized animals.Tibia images are shown here. Mark the desired location of the fracture on the radiograph of the limb to be fractured. Measure from the calcaneal tibial joint to the level of the marked fracture.Calculate the mean fracture length for all trial specimens. On the adjustable fracture device, measure the distance from the outside surface of one support anvil to the center of guillotine impact. Subtract the center of guillotine impact from the fracture length to calculate the fracture positioning jig depth, or JD.Machine or 3D print a U-shaped channel with a height and width equal to the anvil and a depth equal to the JD.Position the specimen in the fracture apparatus in the prone position for femur fractures, or in the supine position for tibia fractures.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.To determine pin length, measure the limb length from the tibial plateau to the level of the posterior malleolus for tibia fractures. To determine pin width, measure the minimum medullary diameter in the fractured limb. Select a needle with a gauge approximately equivalent to the medullary diameter and a length more than 1.5 times pin length.An approximate pin size for a 14-week old C57 black six mouse is 27 gauge, one and a quarter inch, and 22 gauge, one and a half inch for tibia and femur, respectively. Machine or 3D print a gauge with a length equal to pin length minus the needle length. 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.Use an electric clipper or depilatory cream to remove hair from the legs of non-fracture trial specimens from mid-tibia to mid-femur, exposing the knee joint. To pin the tibia, 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. Next, use the gauge and ream until the exposed needle is equal to the gauge length. Retract the needle approximately three millimeters, to provide enough room to cut the needle at the level indicated by the gauge.Crimp 3 millimeters of the distal end of the pin using a pin cutter, and then cut the pin at the level of the gauge. Sync the pin to the articular surface using a rod with a diameter 1.5 times larger than the diameter of the needle. 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.To determine impact depth, measure the diameter of the cortex at the level of the desired fracture on the radiograph. Position a pinned trial specimen in the fracture device using the fracture positioning jig. Rest the impact ram on the uninjured limb.Do not allow the ram to drop. The bone should remain intact during this optimization step. Apply enough downward force on the ram to compress soft tissue, but not fracture the bone.Adjust the impact depth to 75 times cortical diameter to account for the soft tissue. Set the drop height to two centimeters. Position the ram in its starting position by connecting it to the activated electromagnet.Position a trial limb in the fracture apparatus. Press the dorsum of the foot against the fracture positioning jig. Briefly depress the foot switch to release the ram and then reset it to its starting position.Radiograph the impacted trial limb and inspect for any evidence of a fracture. This can be subtle when using low velocities with a controlled impact depth. If no fracture is generated, increase the drop height by two centimeters.If a fracture is generated, record the drop height and multiply it by 1.1. This is the new drop height. Use the new drop height to fracture the next trial limb.Continue the procedure until all trial samples are fractured. Record the final drop height and all parameters from the optimization. Record the trial specimen's age, sex, genotype, and weight.Using an adjustable fracture device and optimized parameters significantly improved the generation of simple transverse fractures. The pre-optimization group only generated a simple transverse fracture in 27 out of 58 samples, or 46.55%of the time. Whereas, the post-optimization group exhibited simple transverse fractures 98.28%of the time.After its development, this technique increased the rigor and the reproducibility of animal models in fracture generation studies. Following the procedure, other techniques, such as micro-CT and histology can be used to answer additional fracture morphology questions.

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