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 (Figure 1A). The metallic surgical instruments must be autoclaved in self-sealing envelopes at 123 &#176;C for 30 min. Disposable items, such as needles and gauze pads, must be procured sterile.
1. Animal preparation
<ol>
	<li>Anesthetize the mouse and perform analgesia in accordance with the veterinary-recommended regimen approved by the&#160;institutional animal care and use program. 
	NOTE: If available, inhalation anesthesia should preferably be performed. A description of the protocol for inhalation anesthesia can be found in the report by Ewald et al.18. However, if the fracture is produced for osteoimmunology studies, this type of anesthesia must be avoided, as evidence shows that several volatile anesthetics, including isoflurane, affect the activity of both innate and adaptive immune cells19,20.</li>
	<li>Once the mouse is immobile, shave the left leg, and then transfer it to the surgical table on a warm heating pad (see Table of Materials) at 37 &#176;C covered with a sterile surgical drape.</li>
	<li>Perform antiseptic washing of the incision area by rubbing the skin with a 10% povidone-iodine sponge. Disinfection should begin along the incision line and extend outward in a circular pattern.&#160;Dry the rubbed area with sterile gauze pads, wash with 70% ethanol, and dry again with a sterile gauze pad. Repeat this procedure three times.</li>
	<li>Place the mouse in the right lateral decubitus position, and immobilize the paws with surgical tape (Figure 1C).</li>
	<li>Drape the mouse so that only the incision region is visible (Figure 1D).</li>
</ol>
2. Surgical procedure
<ol>
	<li>During the surgical procedure, constantly check that the mouse is breathing and provide eye drops in its eyes to avoid dryness and prevent the mouse from becoming blind. 
	NOTE: The whole surgical procedure usually takes ~15-20 min when performed by a trained surgeon. Therefore, applying the eye drops once at the beginning of the procedure should suffice. If the procedure starts to become longer, additional applications can be performed whenever it is identified that the eyes are starting to dry.</li>
	<li>Before proceeding to the incision, evaluate the anesthetic depth by pinching the tail to check the pain response reflex and visually inspecting the rate of breathing (counting the number of thoracic movements per minute)21. Under optimal anesthesia, the mouse should not respond to a tail pinch, and the rate of breathing must be around 55-65 breaths/min21.</li>
	<li>Make a 1 cm cutaneal lateral parapatellar incision with a scalpel blade (Number 11, see Table of Materials), beginning at the level of the tibial tuberosity and extending to the level of the patella and then, for an equal distance, toward the distal femur (Figure 1E).</li>
	<li>With blunt-end scissors, dissect the subcutaneous fascia around the incision line to expose the fascia lata, the lateral vastus, and the femoral biceps muscles22.</li>
	<li>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 (Figure 1F, G).</li>
	<li>Perform a medial luxation of the patella by placing the tip of a straight serrated precision tip tweezer (see Table of Materials) under it and pushing it to the side together with the patellar and quadriceps ligaments, thus exposing the condyles of the femur (Figure 1H).</li>
	<li>Holding the femur with a serrated tip tweezer, flex the knee at 90&#176;, and manually perforate the intramedullary canal of the femur through the intercondylar fossa with a 26 G hypodermic needle (Figure 1I, J).</li>
	<li>Maintaining the knee flexed at 90&#176;, insert a segment of 1.0 cm of a 0.016 in (0.40 mm) stainless steel rod wire (Figure 1K, insert) (see Table of Materials) through the opening into the medullary canal of the femur toward the great trochanter (Figure 1K). 
	NOTE: Maintaining the knee flexed at 90&#176; is crucial for the proper insertion of the wire into the medullary canal. Not doing so will result in the extravasation of the wire out of the bone and surrounding soft tissue lesions.</li>
	<li>Adjust the pre-bent distal extremity of the wire with a straight serrated tip tweezer to tightly fix it in the lateral condyle (Figure 1L). In addition to fixing the wire in place, the bent extremity will facilitate the postmortem removal of the wire.</li>
	<li>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 (Figure 1M).</li>
	<li>Insert a dissecting scissor around the femur diaphysis at an angle of approximately 90&#176;, and gently perform a complete cortical osteotomy (Figure 1N). 
	NOTE: Mice femurs are easily cut. Refrain from applying excessive force during osteotomy to avoid the bending of the intramedullary wire and extensive fracture comminution.</li>
	<li>Reposition the muscles and the patella by pushing the tip of a straight serrated precision tip tweezer over the condyle region.</li>
	<li>Close the muscle fascia with a 6-0 resorbable suture and then the skin using a 6-0 nylon suture (see Table of Materials), both in a simple interrupted fashion (Figure 1O).</li>
	<li>Transfer the mouse to an individual clean cage for recovery. Once awake, the mouse must be able to move freely with unrestricted weight bearing.</li>
	<li>In the following days after surgery, perform analgesia in accordance with the veterinary-recommended regimen approved by the&#160;institutional animal care and use program.</li>
</ol>
3. X-ray imaging
<ol>
	<li>Anesthetize the mouse as described in step 1.1. 
	NOTE: If the radiography is performed right after the surgical procedure and the mouse is still under optimal anesthesia (step 2.2), it is not necessary to perform this step.</li>
	<li>For a clean lateral view of the fractured femur, place the mouse in the dorsal decubitus position, and slightly pull the operated hindlimb to the side.</li>
	<li>Immobilize the paws with surgical tape.</li>
	<li>Perform radiographic imaging according to the available equipment protocol. 
	&#8203;NOTE: For this study, a digital dental X-ray generator was used with the following parameters: 70 kVp voltage, 7 mA current, and 0.2 s exposure time.</li>
</ol>
4. Histology processing and H&#38;E staining
<ol>
	<li>Euthanize the mice with an intraperitoneal overdose of anesthetics (please refer to the veterinary-recommended regimen approved by the&#160;institutional animal care and use program). After checking the depth of anesthesia with a tail pinch, perform cervical dislocation. Next, collect the fractured bone, remove excess surrounding muscle tissue23, and fix the bone in 10% buffered formalin solution (pH 7.4) for 3 days.</li>
	<li>Place the bone samples in labeled histology cassettes (see Table of Materials), and immerse them in 10% EDTA in phosphate-buffered saline (PBS), pH 7.4, for 14 days for decalcification. Change the decalcification solution twice per week.</li>
	<li>Dehydrate the samples in a series of solutions of increasing ethanol concentrations (70%, 80%, 90%, 100%, 100%) for 1 h each.</li>
	<li>Clear the samples in two sequential baths of xylene for 30 min each.</li>
	<li>For wax infiltration, immerse the samples in two sequential paraffin baths at 60 &#176;C for 30 min. Next, embed the samples into blocks for sectioning24. 
	NOTE: To better view the callus, embed the bone with its long axis in the horizontal position to allow for longitudinal cuts.</li>
	<li>Cut the tissue into 4 &#181;m thick sections with a microtome (see Table of Materials).</li>
	<li>Float the sections in a 56 &#176;C water bath, and mount the sections on histological slides (see Table of Materials).</li>
	<li>For H&#38;E staining, deparaffinize the slides in three sequential baths of xylene for 5 min, and rehydrate the tissue in a series of solutions of decreasing ethanol concentrations (95%, 80%, and 70%) for 5 min.</li>
	<li>Rinse the slides in tap water for 30 s, stain the slides with Harris hematoxylin (see Table of Materials) for 6 min, and rinse them in tap water for another 30 s.</li>
	<li>Immerse the slides in 1% hydrochloric acid in ethanol for 30 s and then in 70% ethanol for 30 s.</li>
	<li>Stain with eosin (see Table of Materials) for 2 min, and wash with tap water for 30 s.</li>
	<li>Dehydrate the slides with ethanol (70%, 80%, and 95% for 5 min), and clarify with two baths of xylene for 5 min each.</li>
	<li>For mounting, drip one to two drops of mounting medium (see Table of Materials) onto each slide, and cover the slide with a clean coverslip.</li>
</ol>

<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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+of+fracture+nonunion.">Molecular pathogenesis of fracture nonunion.</a> Journal of Orthopaedic Translation. (14), 45-56 (2018).</li><li>Calori, G. 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=Non-unions.">Non-unions.</a> Clinical Cases in Mineral Bone Metabolism. 14 (2), 186-188 (2017).</li><li>Ekegren, C. L., Edwards, E. R., de Steiger, R., Gabbe, B. 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. T., Dailey, H., Marcucio, R., Hunt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+research+models+in+the+science+of+fracture+healing+-+Pitfalls+&amp;+promises.">Biomedical research models in the science of fracture healing - Pitfalls &amp; promises.</a> Injury. 51 (10), 2118-2128 (2020).</li><li>Schindeler, A., Mills, R. J., Bobyn, J. D., Little, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+for+orthopedic+research+and+bone+tissue+engineering.">Preclinical models for orthopedic research and bone tissue engineering.</a> Journal of Orthopedic Research. 36 (3), 832-840 (2018).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia.">Monitoring of vital signs for long-term survival of mice under anesthesia.</a> Cold Spring Harbor Protocols. 2011 (2), 5563 (2011).</li><li>Stollings, L. 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=Immune+modulation+by+volatile+anesthetics.">Immune modulation by volatile anesthetics.</a> Anesthesiology. 125 (2), 399-411 (2016).</li><li>Sedghi, S., Kutscher, H. L., Davidson, B. A., Knight, P. 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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=Population-based+epidemiology+of+femur+shaft+fractures.">Population-based epidemiology of femur shaft fractures.</a> Journal of Trauma and Acute Care Surgery. 74 (6), 1516-1520 (2013).</li><li>Gunderson, Z. J., Campbell, Z. R., McKinley, T. O., Natoli, R. M., Kacena, M. 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+comprehensive+review+of+mouse+diaphyseal+femur+fracture+models.">A comprehensive review of mouse diaphyseal femur fracture models.</a> Injury. 51 (7), 1439-1447 (2020).</li><li>Haffner-Luntzer, M., Fischer, V., Ignatius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+fracture+healing+between+female+and+male+C57BL/6J+mice.">Differences in fracture healing between female and male C57BL/6J mice.</a> Frontiers in Physiology. 12, 712494 (2021).</li><li>Bonnarens, F., Einhorn, T. 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The authors have no conflicting financial interests.

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">
	<tbody>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		<tr>
			<td>Nonresorbable Nylon Suture thread n&#186; 6</td>
			<td>Atramat</td>
			<td>C1546-NT</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<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>
			<td></td>
		</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...

Watch this Scientific Journal Video about Establishing a Diaphyseal Femur Fracture Model in Mice at JoVE.com

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 ...

establishing-a-diaphyseal-femur-fracture-model-in-mice

Research

JoVE Journal

Biology

1.7K Views.  Federal University of Rio de Janeiro. 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.

Video: Establishing a Diaphyseal Femur Fracture Model in Mice

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37&#176;C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R&#39;s. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...