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 ...

The diaphyseal femur fracture model shown here is a simple and low cost murine model, suited for basic and preclinical studies of long-bone fracture healing. The open surgery technique does not require complex surgical instruments and fixation devices. It allows the visual inspection of the fracture being produced.This model can be used for local transplantation and in vivo testing of therapeutic components, including stem cells, drugs and biomaterials that cannot be delivered by percutaneous, or systemic injection. The surgery protocol requires some knowledge of basic surgery techniques and of the mouse anatomy to perform the critical steps. The visual demonstration in this work can help even for those inexperienced with animal surgery.Demonstrating the procedure will be Leonardo Muller and Bianca Frade, both master students from the Laboratory of Stem Cells and Bone Repair. After transferring the anesthetized animal to the surgical table, disinfect the area to be incised by rubbing the skin with a 10%povidone iodine sponge. Then dry the rubbed area with sterile gauze pads, wash with 70%ethanol and dry again with a sterile gauze pad.Next, place the mouse in the right lateral decubitus position and drape the mouse, making only the incision region visible. Before surgery, place eyedrops in the mouse's eyes to avoid dryness and constantly check the mouse's breathing during the surgical procedure. Make a one centimeter cutaneous lateral parapatellar incision with a scalpel blade number 11, beginning at the level of the tibial tuberosity and extending to the level of the patella, and then for an equal distance towards the distal femur.With blunt end scissors, dissect the subcutaneous fascia around the incision line to expose the fascia lata, the lateral vastus, and the femoral biceps muscles. With the scalpel blade number 11, make another incision in the fascia lata, similar to the one made in the skin, beginning at the level of the tibial tuberosity and running along the biceps femoris aponeurosis until the level of the distal femur to open the articular capsule and access the knee joint. Perform a medial luxation of the patella by placing the tip of a straight serrated precision tip tweezer under it and pushing it to the side together with the patellar and quadriceps ligaments, thus exposing the condyles of the femur.Holding the femur with a serrated tip tweezer, flex the knee at 90 degrees and manually perforate the intramedullary canal of the femur through the intercondylar fossa with a 26-gauge hypodermic needle. Maintaining the knee flexed at 90 degrees, insert a segment of 1.0 centimeters, a 0.016 inch stainless steel rod wire through the opening into the medullary canal of the femur toward the great trochanter. Adjust the prevent distal extremity of the wire with a straight serrated tip tweezer to tightly fix it in the lateral condyle.Next, separate the lateral vastus and femoral biceps muscles through blunt-end dissection with a serrated tip tweezer to access the distal diaphysis of the femur. Insert a dissecting scissor around the femur diaphysis in an angle of approximately 90 degrees, and gently perform a complete cortical osteotomy. Reposition the muscles and the patella by pushing the tip of a straight serrated precision tip tweezer over the condyle region.Close the muscle fascia with a 6-0 reabsorbable suture, and then the skin using a 6-0 nylon suture, both in simple interrupted fashion. The radiographs of acceptable fracture patterns show transverse diaphyseal fractures in which the fracture lines are at a 90 degree angle to the axis of the bone, short oblique fractures in which the fracture line is less than 30 degrees relative to the axis of the bone, reducible fragmentary fractures, where a few small fragments of bone are seen, but the anatomical alignment of the bone remains. Representative radiographs of incorrectly placed wires are shown here where the wire is not inside the medullary canal of the proximal femur fragment, thus resulting in incorrect fixation of the fractured bone, and a case where the wire did not pass through any bone fragment and the fractured bone is completely unaligned.Visible callus at the fracture site on day 14 and day 21 after surgery indicates the regenerative process of the model. Histological analysis shows the evaluation of the callus at the fracture site. The callus, initially on day seven, presents extensive areas of highline cartilage around the fracture line.On day 14, ossification fronts are observed around the cartilage area, forming trabecular bone and cavities filled with reconstituted bone marrow. Finally, on day 21, the cartilage areas are almost completely replaced by trabecular bone, indicating successful bone bridging. The success of the model depends on the correct insertion of the wire.Therefore, it's critical to keep the knee flexed at a 90 degree angle during this step. Additionally, micro-computed demography can be used to evaluate cell's evolution and the characteristics of the bone being formed in a tri-dimensional and quantitative scale.

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