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>

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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 and therapeutic protocols16,17. 
In the mouse, both the femur and the tibia can be used for long bone fracture healing studies. In this model, the femur was chosen instead of the tibia because it is a straight bone with a larger diameter and better soft tissue coverage. On the other hand, the diaphysis of the mouse tibia is curved, and its caliber progressively decreases along the distal end, which complicates the insertion of intramedullary fixation devices26. Therefore, the characteristics of the femur make it ideal for models in which intramedullary fixation is intended. Regarding the sex, male mice were used, as there is evidence that males show faster fracture healing with more prominent cartilaginous callus formation compared to females27. However, if necessary, the technique can be easily adapted to females by simply adjusting the size of the intramedullary wire to fit the slightly smaller length of the female femur.
Compared to closed fracture models that make use of the three-point bending mechanism with the guillotine28, the open surgery model described here is also advantageous because it exposes the fracture site, which allows the researcher to visually see the fracture being produced. This visualization helps to avoid technical errors that result in the following unacceptable fracture patterns: severe fragment displacement, which does not allow the anatomical realignment of the bone (nonreducible fractures); extensive fragmentation of the bone into several small pieces (comminution), a condition that may impair the repair process; and/or the misplacement of the fixation devices. As the fracture is caused by gentle osteotomy in this model, extensive fragment displacement and/or comminution are generally not observed. 
However, the technique is limited in the sense that it requires greater technical surgical skills and knowledge of the anatomy of the mouse than other methods. In addition, the small size of the mouse makes manipulation trickier compared to rats or large animal models. Once these limitations are overcome with training, the rate of success in the production of acceptable fractures is nearly 100%, reducing the number of animal removals from the study.
Furthermore, the open surgery fracture model allows the local application of therapeutic agents, such as stem/progenitor cells, biomaterials, and/or pharmaceutical drugs, which would not be possible to apply using percutaneous or systemic delivery26. Finally, fixation with intramedullary devices is easier, cheaper, and more customizable than with plate and external devices and mimics the most commonly used clinical strategy for the treatment of long bone fractures29. Therefore, the model described here represents a low-cost model for studying fracture healing, both in basic and translational settings, meaning this study contributes not only to increased knowledge of fracture healing biology but also to the development of new therapeutic strategies for bone repair.

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><tbody><tr><td>Alcohol 70&amp;ordm;</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.&amp;nbsp;</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&amp;nbsp;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&amp;nbsp;sponge</td><td>Rioqu&amp;iacute;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&amp;nbsp;</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&amp;ordm; 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&amp;nbsp;</td><td>Lonza</td><td>&amp;nbsp;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&amp;ordm; 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&quot;</td><td>Orthometric</td><td>56.50.2016</td><td></td></tr><tr><td>Scalpel n&amp;ordm; 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&amp;nbsp;</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 angle of 90&#176; to the axis of the bone), oblique (curved or sloped pattern without fragment displacement), or short oblique (around 30&#176; relative to the axis of the bone) (Figure 2A-D). These patterns are acceptable because they all will progress to repair through endochondral bone formation (i.e, with callus formation) if the bone fragments are properly aligned (reduced), thus achieving the main objective of the model.&#160;Therefore, unacceptable fractures are only those with extensive comminution (multiple small bone fragments), with shortening of the limb as a consequence of poor alignment, and with misplaced wires (Figure 3). Animals with unacceptable fracture patterns must be excluded from the study. With time, a robust and visible callus should be observed at the fracture site (Figure 4).
In addition, a histological examination can be performed at 7 days, 14 days, and 21 days after fracture to assess tissue neoformation within the fractured area. As fixation with intramedullary wires allows for a certain degree of movement of the bone fragments, the regenerative process follows the endochondral mechanism of ossification, in which robust areas of hyaline cartilage are seen around the fracture line on day 7 (Figure 5A,B). On day 14, ossification fronts are observed around the cartilage area, forming trabecular bone and cavities filled with reconstituted bone marrow (Figure 5C,D). Finally, on day 21, the cartilage areas are almost completely replaced by trabecular bone, indicating successful bony bridging (Figure 5E,F) and the validity of the model for fracture healing studies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64766/64766fig01.jpg" /> 
Figure 1: Photomicrographs illustrating the steps of the surgical procedure to produce diaphyseal femur fractures fixed with an intramedullary wire in the mouse. (A) Organization of the sterile surgical instruments on the operating table. (B) Intraperitoneal injection of the anesthetics. (C) Positioning of the mouse in the lateral decubitus position and immobilization of the paws. (D) Draping of the mouse, leaving exposed only the area that will be operated on. (E) The cutaneous lateral parapatellar incision. (F,G) Views of the fascia lata incision. (H) Medial luxation of the patella, exposing the region of the femoral condyle. (I) Positioning of the needle in the intercondylar fossa. (J) Perforation of the femoral medullary canal. (K) Insertion of the intramedullary wire through the femoral opening. (L) Adjustment of the bent extremity of the wire in the lateral condyle. (M) Blunt-end separation of the surrounding muscles. (N) Full cortical femoral osteotomy. (O) Closure of the soft tissues. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64766/64766fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64766/64766fig02.jpg" /> 
Figure 2: Representative radiographs of acceptable fracture patterns. (A,B) Transverse diaphyseal fractures (the fracture lines are at a 90&#176; angle to the axis of the bone). (C) Short oblique fracture (the fracture line is less than 30&#176; relative to the axis of the bone). (D) Reducible fragmentary fracture (few small fragments of bone are seen, but the anatomical alignment of the bone remains). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64766/64766fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64766/64766fig03.jpg" /> 
Figure 3: Representative radiographs of incorrectly placed wires. (A) In this mouse, the wire is not inside the medullary canal of the proximal femur fragment, thus resulting in incorrect fixation of the fractured bone.(B) In this case, the wire did not pass through any bone fragment, and the fractured bone is completely unaligned. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64766/64766fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64766/64766fig04.jpg" /> 
Figure 4: Visible callus at the fracture site. Representative radiographs of fracture calluses on (A) day 14 and (B) day 21 after surgery, showing that the regenerative process of the model follows the indirect (endochondral) pathway. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64766/64766fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64766/64766fig05.jpg" /> 
Figure 5: Histological analysis of the fracture calluses. Representative images of fractured bones on (A,B) day 7, (C,D) day 14, and (E,F) day 21 after surgery stained with H &#38; E. Note the evolution of the callus; the callus initially presents with extensive areas of hyaline cartilage around the fracture line (insert in A, magnified in B), these areas then serve as templates for the formation of trabecular bone (insert in C, magnified in D), and the process culminates in the complete replacement of the cartilage by bone and, thus, bone bridging (insert in E, magnified in F). Scale bars: (A,C,D) 500 &#181;m; (B,D,F) 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64766/64766fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</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

Nanyang Technological University

Research

JoVE Journal

Biology

1.9K Views.  Federal University of Rio de Janeiro. Het hier getoonde diafysaire femurfractuurmodel is een eenvoudig en goedkoop muizenmodel, geschikt voor basis- en preklinische studies van genezing van longbonefracturen. De open chirurgietechniek vereist geen complexe chirurgische instrumenten en fixatieapparaten. Het maakt de visuele inspectie van de geproduceerde breuk mogelijk.Dit model kan worden gebruikt voor lokale transplantatie en in vivo testen van therapeutische componenten, waaronder stamcellen, geneesmiddelen en biomateria...

Video: Establishing a Diaphyseal Femur Fracture Model in Mice

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

Medicine

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Cantilever Bending of Murine Femoral Necks

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20&#176; relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6&#176; &plusmn; 1.5&#176;, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and ...

Bioengineering

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue ...

Neuroscience

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

Establishing a Diaphyseal Femur Fracture Model in Mice

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