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.

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

Research

JoVE Journal

Medicine

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

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

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

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

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

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

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

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 ...