Name: establishing a diaphyseal femur fracture model in mice
Uploaded: 2022-12-09T14:30:00
Description: Watch this Scientific Journal Video about Establishing a Diaphyseal Femur Fracture Model in Mice at JoVE.com

This work was funded by the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ).

All the experiments were approved by the Animal Use and Care Committee of the Center for Health Sciences of the Federal University of Rio de Janeiro (Protocol Number 101/21). Male Balb/c mice at 10-12 weeks of age (25-30 g body weight) were used in this study. The surgical procedure takes approximately 15-20 min per mouse. Before each procedure, the required instruments (listed in the Table of Materials) must be organized over a sterile surgical field covering the operating table (Fi...

<ol>
	<li>Florencio-Silva, R., Sasso, G. R., Sasso-Cerri, E., Simoes, M. J., Cerri, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+bone+tissue:+Structure,+function,+and+factors+that+influence+bone+cells.">Biology of bone tissue: Structure, function, and factors that influence bone cells.</a> BioMed Research International. 2015, 421746 (2015).</li><li>Bahney, 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=Cellular+biology+of+fracture+healing.">Cellular biology of fracture healing.</a> Journal of Orthopedic Research. 37 (1), 35-50 (2019).</li><li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+Mechanisms+and+interventions.">Fracture healing: Mechanisms and interventions.</a> Nature Reviews Rheumatology. 11 (1), 45-54 (2015).</li><li>Perren, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+Fracture+healing+understood+as+the+result+of+a+fascinating+cascade+of+physical+and+biological+interactions.+Part+II.">Fracture healing: Fracture healing understood as the result of a fascinating cascade of physical and biological interactions. Part II.</a> Acta Chirurgiae Orthopaedicae et Traumatologiae Cechoslovaca. 82 (1), 13-21 (2015).</li><li>Giannoudis, P. V., Krettek, C., Lowenberg, D. W., Tosounidis, T., Borrelli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing+adjuncts-The+world's+perspective+on+what+works.">Fracture healing adjuncts-The world's perspective on what works.</a> Journal of Orthopaedic Trauma. 32, 43-47 (2018).</li><li>Kates, S. 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=Outside+the+bone:+What+is+happening+systemically+to+influence+fracture+healing.">Outside the bone: What is happening systemically to influence fracture healing.</a> Journal of Orthopaedic Trauma. 32, 33-36 (2018).</li><li>Ding, Z. C., Lin, Y. K., Gan, Y. K., Tang, T. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence,+costs+and+predictors+of+non-union,+delayed+union+and+mal-union+following+long+bone+fracture.">Incidence, costs and predictors of non-union, delayed union and mal-union following long bone fracture.</a> Internation Journal of Environmental Research and Public Health. 15 (12), 2845 (2018).</li><li>Aziziyeh, R., 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+burden+of+osteoporosis+in+four+Latin+American+countries:+Brazil,+Mexico,+Colombia,+and+Argentina.">The burden of osteoporosis in four Latin American countries: Brazil, Mexico, Colombia, and Argentina.</a> Journal of Medical Economics. 22 (7), 638-644 (2019).</li><li>Kostenuik, P., Mirza, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing+physiology+and+the+quest+for+therapies+for+delayed+healing+and+nonunion.">Fracture healing physiology and the quest for therapies for delayed healing and nonunion.</a> Journal of Orthopaedic Research. 35 (2), 213-223 (2017).</li><li>Gomez-Barrena, 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=fracture+healing:+cell+therapy+in+delayed+unions+and+nonunions.">fracture healing: cell therapy in delayed unions and nonunions.</a> Bone. 70, 93-101 (2015).</li><li>Schlundt, 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=Clinical+and+research+approaches+to+treat+non-union+fracture.">Clinical and research approaches to treat non-union fracture.</a> Current Osteoporosis Reports. 16 (2), 155-168 (2018).</li><li>Gomez-Barrena, 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=Feasibility+and+safety+of+treating+non-unions+in+tibia,+femur+and+humerus+with+autologous,+expanded,+bone+marrow-derived+mesenchymal+stromal+cells+associated+with+biphasic+calcium+phosphate+biomaterials+in+a+multicentric,+non-comparative+trial.">Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial.</a> Biomaterials. 196, 100-108 (2018).</li><li>Ryan, G., 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=Systemically+impaired+fracture+healing+in+small+animal+research:+A+review+of+fracture+repair+models.">Systemically impaired fracture healing in small animal research: A review of fracture repair models.</a> Journal of Orthopedic Research. 39 (7), 1359-1367 (2021).</li><li>Marmor, M. 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As the number of fractures increases worldwide9,10,25, innovative treatments for nonunion are becoming increasingly urgent. As fracture healing involves a complex and tightly orchestrated summation of events that occur over a long timescale3, the use of valid animal models is central to improving our understanding of the mechanisms that determine the success of bone repair and to selecting effective drugs...

The skeleton is a vital and functionally versatile organ. The bones of the skeleton enable body posture and movement, protect the internal organs, produce hormones that integrate physiological responses, and are the site of hematopoiesis and mineral storage1. If fractured, bones have a remarkable capacity to regenerate and fully restore their pre-injury form and function. The healing process begins with the formation of a hematoma and an inflammatory response, which induces the activation and condensation of skeletal stem/progenitor cells from the periosteum, endosteum, and bone marrow and their subsequent differentiation to form the soft cartilaginous callus. The bridging of the fractured ends then occurs through a process that resembles endochondral bone formation, in which the cartilaginous scaffold expands and then mineralizes, forming the hard osseous callus. Finally, the hard callus is gradually remodeled by osteoclasts and osteoblasts to restore the original bone structure2,3.
Although the fracture healing process is fairly robust, it involves an intricate summation of events and is significantly influenced by several individual factors, including the general health status, age, and sex of the patient, as well as injury factors, such as the mode of mechanical stabilization of the fractured bone, the occurrence of infection, and the severity of the surrounding soft tissue injury4,5,6. Therefore, failures are common, leading to the development of nonunion, which greatly impacts patient rehabilitation and quality of life7,8. Due to the increasing number of fractures as a result of high-energy trauma and aging, as well as the high costs of treatments, nonunion fractures have become a burden for health systems worldwide9,10. This increasing burden highlights the urgent need for innovative therapeutic strategies to improve bone repair11,12 based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials13,14.
In pursuit of this goal, animal models have been widely used in studies aiming to understand the fundamental biology of fracture healing mechanisms and in proof-of-concept preclinical studies aiming to devise new therapeutic strategies to promote bone repair15,16,17. Small-animal models, such as the mouse, are excellent for fracture healing studies because of the wide availability of genetically modified strains and reagents for experimental analyses and their low maintenance costs. Additionally, mice have a rapid healing time course, which allows for the temporal analysis of all stages of the repair process15. However, the small size of the animal can pose challenges for the surgical production of fractures with fixation modes similar to those applied in humans. This protocol describes a simple and low-cost model of fracture healing in mice using an open femoral osteotomy stabilized with an intramedullary wire, which resembles the most common bone repair process, through cartilaginous callus formation, and can be used both in basic and translational investigations in which access to the fracture site is required.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Alcohol 70&#186;</td>
			<td>Merck</td>
			<td>109-56-8</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Canada balsam (mounting medium)</td>
			<td>Merck</td>
			<td>C1795</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Cefazoline</td>
			<td>ABL</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Merck</td>
			<td>CSL284525</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Dental X-Ray Generator</td>
			<td>Focus</td>
			<td>-</td>
			<td>Sold by Instrumentarium Dental Inc.&#160;</td>
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			<td>DEPC water</td>
			<td>Merck</td>
			<td>W4502</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Dissecting Scissor</td>
			<td>ABC Instrumentos</td>
			<td>0327</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Vetec</td>
			<td>60REAVET014340</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Eosin solution</td>
			<td>Laborclin</td>
			<td>EA-65</td>
			<td>Similar brands of the item may be used according to local availability</td>
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			<td>Ethanol P.A</td>
			<td>Vetec</td>
			<td>60REAVET012053</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Gauze&#160;pads</td>
			<td>Cremer</td>
			<td>Not applicable</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Harris Hematoxylin Solution</td>
			<td>Laborclin</td>
			<td>620503</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Tonkey Electrical Technology</td>
			<td>E114273</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Histological slides</td>
			<td>Merck</td>
			<td>CSL294875X25</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Histology cassettes</td>
			<td>Merck</td>
			<td>H0542-1CS</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Hydrochloric acid - 37%</td>
			<td>Merck</td>
			<td>258148</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>324918</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Iodopovidone&#160;sponge</td>
			<td>Rioqu&#237;mica</td>
			<td>372106</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Ceva</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Lacribel collyrium</td>
			<td>Cristalia</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>149AUTO00C1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Tooth Forceps Tweezer</td>
			<td>ABC Instrumentos</td>
			<td>0164</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Needle 26 G</td>
			<td>BD</td>
			<td>2239</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Needle Holder&#160;</td>
			<td>Golgran</td>
			<td>135-18</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
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			<td>Nonresorbable Nylon Suture thread n&#186; 6</td>
			<td>Atramat</td>
			<td>C1546-NT</td>
			<td>Or any general available supplier</td>
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			<td>Paraffin</td>
			<td>Exodo</td>
			<td>8002 - 74 - 2</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>30525-89-4</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>PBS 1x&#160;</td>
			<td>Lonza</td>
			<td>&#160;BE17-516F</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Resorbable Nylon Suture thread n&#186; 6</td>
			<td>Atramat</td>
			<td>C1596-45B</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Rod Wire SS CrNi 0.016&#34;</td>
			<td>Orthometric</td>
			<td>56.50.2016</td>
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		</tr>
		<tr>
			<td>Scalpel n&#186; 11</td>
			<td>Descarpak</td>
			<td>15782</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Serrated Tip Tweezer</td>
			<td>Quinelato</td>
			<td>QC.404.12</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Phillips</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M</td>
			<td>2734</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Surgical tnt field</td>
			<td>Polarfix</td>
			<td>6153</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Tramadol hydrochloride</td>
			<td>Teuto&#160;</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Water bath for histology</td>
			<td>Leica</td>
			<td>HI1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride</td>
			<td>Ceva</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Dinamica</td>
			<td>60READIN001105</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
	</tbody>
</table>

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.

The most simple and immediate way to evaluate the success of the surgical procedure in producing the fracture is X-ray imaging. Radiographs can be performed immediately after surgery, with the mouse still under anesthesia, and subsequently 7 days, 14 days, and 21 days after the fracture to evaluate the callus formation and progression. Acceptable fracture patterns are those in which the cortices are fully ruptured, the wires are correctly placed within the medullary canal, and the fracture lines are transverse (with an a...

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Establishing a Diaphyseal Femur Fracture Model in Mice

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 ...

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