This work was supported by grants from the Department of Defense (DoD) US Army Medical Research and Materiel Command (USAMRMC) Congressionally Directed Medical Research Programs (CDMRP) (PR121604) and the National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH R01 AR068332 to&#160;Uma Sankar.&#160;Justin Williams is supported through a Comprehensive Musculoskeletal T32 Training Program from NIAMS/NIH (AR065971).

The following procedure was performed with approval from the Indiana University School of Medicine Institutional Animal Care and Use Committee (IACUC). All survival surgeries were performed under sterile conditions as outlined by the NIH guidelines. Pain and risk of infections were managed with proper analgesics and antibiotics to ensure a successful outcome.
1. Anesthesia and Preparation
<ol>
	<li>Weigh the mouse and anesthetize it with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) administered via the intraperitoneal (I.P.) route. Place the mouse in an empty cage and monitor it until it is fully sedated.</li>
	<li>Ensure that the mouse is sedated using a toe pinch reflex. Apply ophthalmic ointment to its eyes to protect them from drying out.</li>
	<li>Remove the fur from the right limb. Wipe down the surgical site with an iodine-based scrub and 70% ethanol. Scrub the surgical site starting at the center of the knee and making a circular sweep outward. Repeat this 3x with fresh scrubs, ending with 70% ethanol.</li>
	<li>Administer a preoperative dose of buprenorphine hydrochloride analgesia (0.03 mg/kg) subcutaneously for the immediate postoperative pain management.</li>
	<li>Place the mouse on a heating pad covered by a sterile surgical pad.</li>
</ol>
2. Surgical Approach
NOTE: Prior to the fracture, the weight and the drop height should be empirically determined for the specific strain, age, and sex of the mice prior to the surgery. This surgical procedure is optimized for C57BJ6 male mice at 10 weeks of age.
<ol>
	<li>Place the mouse on its back and flex the knee of the operative leg. Using a scalpel blade, make a 1.5 cm incision centered over the knee joint.</li>
	<li>Laterally displace the patella using forceps to expose the distal end of the femur. Insert a 1.5-in long 25-gauge stainless steel hypodermic needle at the center of the trochlear groove, down the length of the medullary canal in a retrograde manner and through the proximal end of the femur. Take an X-ray to ensure the proper placement of the pin. 
	NOTE: The needle should exit the dorsal side of the mouse to create a path for the guidewire.</li>
	<li>Pass a 4-in long 36-gauge tungsten guidewire through the shaft of the needle, entering through the hub at the distal femur and exiting the bevel on the dorsal side of the mouse.</li>
	<li>Following the successful placement of the guidewire, carefully remove the 25-gauge needle by gently pulling on the hub while holding the limb and the guidewire in place. Confirm the placement of the guidewire by X-ray.</li>
	<li>Hold a 391 g weight from a height of 34.6 cm above the impact disc (Figure 1A). Position the femur horizontally across the two supporting points, such that the intertrochanteric and supracondylar regions of the femur rest on the support anvils (Figure 1B) and the lateral side of the limb is facing the loading point (Figure 1C). Drop the weight and carefully remove the mouse from the device immediately following the fracture.</li>
	<li>Confirm the fracture location by X-ray.</li>
	<li>Insert the 24-gauge stainless steel hypodermic tubing over the guidewire to stabilize the fractured femur. 
	NOTE: This application may require some force as the point of entry was generated using a needle of a smaller diameter. This difference in diameter effectively prevents a potential migration of the 24-gauge rod through the proximal end of the femur. The depth of insertion can be felt manually as the blunt tubing meets the cortical bone of the greater trochanter.</li>
	<li>Confirm the position of the steel rod and the stabilization of the fractured femur by X-ray before removing the guidewire.</li>
	<li>Cut the excess tubing at the distal end of the femur using wire cutters. Bury the exposed tubing under the surface of the condyles using forceps to apply a gentle downward force, being careful not to dislocate the knee joint.</li>
	<li>Reposition the patella using forceps. Close the incision site with a 5-0 absorbable suture.</li>
</ol>
3. Postoperative Management
<ol>
	<li>Following surgery, the mice may be injected with up to 500 &#181;L of sterile saline via the I.P. route to aid them in their postoperative recovery.</li>
	<li>Monitor the animals on a heated recovery bed until they awaken from surgery. Once ambulatory, return them to their cage.</li>
	<li>Continue to closely monitor the mice for several days after the surgery to ensure they are healing properly and regain mobility. Administer buprenorphine hydrochloride analgesia (0.03 mg/kg) subcutaneously every 6&#160;h for 3 days following the surgery, and as needed thereafter. Avoid the use of non-steroidal anti-inflammatory drugs (NSAIDs) as they have been shown to impair bone healing after surgery.</li>
</ol>

<ol>
	<li>Schnell, S., Friedman, S. M., Mendelson, D. A., Bingham, K. W., Kates, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+1-Year+Mortality+of+Patients+Treated+in+a+Hip+Fracture+Program+for+Elders.">The 1-Year Mortality of Patients Treated in a Hip Fracture Program for Elders.</a> Geriatric Orthopaedic Surgery &amp; Rehabilitation. 1 (1), 6-14 (2010).</li><li>Burge, 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=Incidence+and+economic+burden+of+osteoporosis-related+fractures+in+the+United+States,+2005-2025.">Incidence and economic burden of osteoporosis-related fractures in the United States, 2005-2025.</a> Journal of Bone and Mineral Research. 22 (3), 465-475 (2007).</li><li>Cunningham, B. P., Brazina, S., Morshed, S., Miclau, T. <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:+A+review+of+clinical,+imaging+and+laboratory+diagnostic+options.">Fracture healing: A review of clinical, imaging and laboratory diagnostic options.</a> Injury. 48, S69-S75 (2017).</li><li>Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+an+anti-fracture+agent+heal+fractures?.">Can an anti-fracture agent heal fractures?.</a> Clinical Cases in Mineral and Bone Metabolism. 7 (1), 11-14 (2010).</li><li>Hak, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+union+and+nonunions:+epidemiology,+clinical+issues,+and+financial+aspects.">Delayed union and nonunions: epidemiology, clinical issues, and financial aspects.</a> Injury. 45, S3-S7 (2014).</li><li>Decker, S., Reifenrath, J., Omar, M., Krettek, C., Muller, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-osteotomy+and+osteotomy+large+animal+fracture+models+in+orthopedic+trauma+research.">Non-osteotomy and osteotomy large animal fracture models in orthopedic trauma research.</a> Orthopaedic Reviews (Pavia). 6 (4), 5575 (2014).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+bone+healing+models:+standards,+tips,+and+pitfalls+results+of+a+consensus+meeting.">Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting.</a> Bone. 49 (4), 591-599 (2011).</li><li>Jacenko, O., Olsen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mouse+models+in+studies+of+skeletal+disorders.">Transgenic mouse models in studies of skeletal disorders.</a> Journal of Rheumatology Supplement. 43, 39-41 (1995).</li><li>Nikolaou, V. S., Efstathopoulos, N., Kontakis, G., Kanakaris, N. K., Giannoudis, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+osteoporosis+in+femoral+fracture+healing+time.">The influence of osteoporosis in femoral fracture healing time.</a> Injury. 40 (6), 663-668 (2009).</li><li>Bain, S. D., Bailey, M. C., Celino, D. L., Lantry, M. M., Edwards, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-dose+estrogen+inhibits+bone+resorption+and+stimulates+bone+formation+in+the+ovariectomized+mouse.">High-dose estrogen inhibits bone resorption and stimulates bone formation in the ovariectomized mouse.</a> Journal of Bone and Mineral Research. 8 (4), 435-442 (1993).</li><li>Haffner-Luntzer, M., Kovtun, A., Rapp, A. E., 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=Mouse+Models+in+Bone+Fracture+Healing+Research.">Mouse Models in Bone Fracture Healing Research.</a> Current Molecular Biology Reports. 2 (2), 101-111 (2016).</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 in Rheumatology. 11 (1), 45-54 (2015).</li><li>Schindeler, A., McDonald, M. M., Bokko, P., 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=Bone+remodeling+during+fracture+repair:+The+cellular+picture.">Bone remodeling during fracture repair: The cellular picture.</a> Seminar in Cellular and Developmental Biology. 19 (5), 459-466 (2008).</li><li>Ai-Aql, Z. S., Alagl, A. S., Graves, D. T., Gerstenfeld, L. C., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+controlling+bone+formation+during+fracture+healing+and+distraction+osteogenesis.">Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis.</a> Journal of Dental Research. 87 (2), 107-118 (2008).</li><li>Gerstenfeld, L. 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=Three-dimensional+Reconstruction+of+Fracture+Callus+Morphogenesis.">Three-dimensional Reconstruction of Fracture Callus Morphogenesis.</a> Journal of Histochemistry &amp; Cytochemistry. 54 (11), 1215-1228 (2006).</li><li>Marsell, R., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+bone+healing+therapies.">Emerging bone healing therapies.</a> Journal of Orthopaedic Trauma. 24, S4-S8 (2010).</li><li>Lybrand, K., Bragdon, B., Gerstenfeld, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+bone+healing:+fracture,+marrow+ablation,+and+distraction+osteogenesis.">Mouse models of bone healing: fracture, marrow ablation, and distraction osteogenesis.</a> Current Protocols of Mouse Biology. 5 (1), 35-49 (2015).</li><li>Garcia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LockingMouseNail--a+new+implant+for+standardized+stable+osteosynthesis+in+mice.">The LockingMouseNail--a new implant for standardized stable osteosynthesis in mice.</a> Journal of Surgical Research. 169 (2), 220-226 (2011).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internal+locking+plate+to+study+intramembranous+bone+healing+in+a+mouse+femur+fracture+model.">An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.</a> Journal of Orthopaedic Research. 28 (3), 397-402 (2010).</li><li>Garcia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+internal+fixation+of+femoral+fractures+in+mice:+impact+of+stability+on+fracture+healing.">A new technique for internal fixation of femoral fractures in mice: impact of stability on fracture healing.</a> Journal of Biomechistry. 41 (8), 1689-1696 (2008).</li><li>Holstein, J. H., 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=Advances+in+the+establishment+of+defined+mouse+models+for+the+study+of+fracture+healing+and+bone+regeneration.">Advances in the establishment of defined mouse models for the study of fracture healing and bone regeneration.</a> Journal of Orthopaedic Trauma. 23 (5 Suppl), S31-S38 (2009).</li><li>Bonnarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+closed+fracture+in+laboratory+animal+bone.">Production of a standard closed fracture in laboratory animal bone.</a> Journal of Orthopaedic Research. 2 (1), 97-101 (1984).</li><li>Holstein, J. H., Menger, M. D., Culemann, U., Meier, C., Pohlemann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+locking+femur+nail+for+mice.">Development of a locking femur nail for mice.</a> Journal of Biomechistry. 40 (1), 215-219 (2007).</li><li>McBride-Gagyi, S. H., McKenzie, J. A., Buettmann, E. G., Gardner, M. J., Silva, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp2+conditional+knockout+in+osteoblasts+and+endothelial+cells+does+not+impair+bone+formation+after+injury+or+mechanical+loading+in+adult+mice.">Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice.</a> Bone. 81, 533-543 (2015).</li><li>Williams, J. N., 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=Inhibition+of+CaMKK2+Enhances+Fracture+Healing+by+Stimulating+Indian+Hedgehog+Signaling+and+Accelerating+Endochondral+Ossification.">Inhibition of CaMKK2 Enhances Fracture Healing by Stimulating Indian Hedgehog Signaling and Accelerating Endochondral Ossification.</a> Journal of Bone and Mineral Research. , (2018).</li></ol>

The authors of this manuscript have nothing to disclose. The authors further state that there are no restrictions on the full access to all the material used in the study reported in this manuscript.

The goal of this surgical procedure is to generate standardized closed femoral fractures in mice. A key advantage of this model is that the internal fixation takes place after the generation of the fracture, thereby avoiding an angulation of the intramedullary rod. Perhaps the most critical aspect of this protocol is the generation of a standardized transverse fracture at the femoral midshaft, as the fracture geometry is dependent on the applied bending force and the positioning of the hind limb. Improper positioning of ...

Fractures are among the most common injuries occurring to the musculoskeletal system and are associated with a tremendous socioeconomic burden, including treatment costs that are projected to surpass $25 billion annually in the United States1,2. Although the majority of fractures heal without incident, healing is associated with substantial downtime and loss of productivity. Approximately 5 - 10% of all fractures result in a delayed healing or&#160;non-union, due to age or other underlying chronic health conditions, such as osteoporosis and&#160;diabetes mellitus3,4,5. No FDA-approved pharmacological treatments are currently available to promote efficient bone healing and shorten recovery time.
Fracture healing is a complex and highly dynamic process involving the coordination of multiple cell types. Hence, a comprehensive understanding of the cellular and molecular events associated with bone regeneration is crucial to the identification of therapeutic targets that accelerate this process. As with other human diseases, the establishment of a highly amenable and reproducible animal model is crucial in the study of bone healing. Larger animals, such as sheep and swine, have bone remodeling properties and biomechanics similar to humans, but are expensive, require substantial healing time, and are not readily amenable to genetic manipulation6. On the other hand, small animal models, such as rats and mice, offer many advantages, including an ease of handling, low costs of maintenance, short breeding cycles, and a shorter healing time7. Furthermore, the mouse genome is fully sequenced, allowing for the rapid manipulation and generation of genetic variants. Thus, the mouse is a powerful model system to study human disease, injury, and repair8. In humans, comorbidities like osteoporosis and diabetes mellitus increase the likelihood of a delayed healing. A number of existing mouse models are available to study the effects of comorbidities such as osteoporosis and diabetes mellitus on bone injury and healing. Patients suffering from osteoporosis have a markedly decreased bone formation during the later stages of a fracture healing9. Ovariectomized (OVX) mice exhibit rapid bone loss and delayed bone healing similar to that observed in postmenopausal osteoporosis10,11. Additionally, many mouse models of type I and type II diabetes mimic the low bone mass phenotypes and impaired fracture healing seen in humans11. Moreover, murine fracture models serve as a versatile platform to study the complex biological processes occurring in the callus and explore novel therapeutic strategies that accelerate bone tissue regeneration.
Despite differences in bone structure and metabolism, the overall process of bone fracture healing remains very similar in mice and humans, involving a combination of endochondral and intramembranous ossification followed by bone remodeling. Endochondral ossification involves the recruitment of progenitor cells to less mechanically stable regions surrounding the fracture gap, where they differentiate into chondrocytes that hypertrophy and mineralize the cartilage to produce a soft callus. The second wave of progenitor cells infiltrate the callus and differentiate into mature osteoblasts that secrete new bone matrix12,13,14,15. During intramembranous ossification, progenitors on the periosteal and endosteal surfaces directly differentiate into matrix secreting osteoblasts and facilitate the bridging of the fracture gap9,11,12,13. Together, the endochondral and intramembranous ossifications result in the development of a hard callus, which is further remodeled over time to form a strong secondary bone capable of supporting mechanical loads13,14,15. In healthy humans, the healing process takes approximately 3 months, compared to only 35 days in mice16.
Fracture healing has commonly been studied using either open or closed surgical models17. Open surgical approaches, such as the generation of a critically sized defect or complete osteotomy, standardize the injury location and geometry to reduce deviations caused by comminuted fractures. Osteotomies serve as an excellent model to study the underlying mechanism behind a non-union because healing is often delayed compared to closed fractures. Furthermore, a rigid external fixation is required to stabilize the osteotomized bone, meaning the regeneration will primarily depend on the intramembranous ossification. Open surgical approaches use devices such as locking nails, pin-clips, and locking plates to provide axial and rotational stability to the fractured limb; however, such devices are expensive and require significantly more time in surgery18,19,20,21. On the other hand, closed models are stabilized with a simple intramedullary fixation device, allowing for enough instability to stimulate endochondral healing. As a result, closed fracture models do not readily mimic the conditions of a non-union. Internal fixation techniques, such as intramedullary pins, nails, and compression screws, are advantageous as they are cheap, easy to use, and minimize the time in surgery21,22,23. In some cases, intramedullary pins are inserted prior to the fracture, but the bending of the intramedullary pin can lead to the angulation or displacement of the fractured femur, contributing to a variable callus size and healing. The fracture location and geometry are more difficult to standardize in closed models, as they are generated using a three-point bending device, wherein a weight is dropped on the diaphysis. However, with the proper technique, this surgical approach offers rapid and consistent results. Moreover, the closed fracture model serves as a clinically relevant tool to study fractures caused by high force impact or mechanical stress22.
This surgical protocol was adapted from previously described methods using an intramedullary pin to stabilize fractured femurs in rats and mice22,24,25. First, an intramedullary needle of a small diameter is inserted through the intracondylar notch to establish a point of entry, and a guidewire is introduced prior to generating a transverse fracture at the femoral midshaft using a gravity-dependent three-point bending device. Following the successful generation of a closed femoral fracture, an intramedullary rod of a larger diameter is incorporated over the guide wire to stabilize the fractured femur. This method avoids the risk of delayed healing caused by the angulation of the intramedullary pin during the fracture, as the placement of the rod post-fracture allows for the repositioning and optimized stabilization of the injured femur.

<table><tbody><tr><td>Oster Minimax Trimmer</td><td>Animal World Network</td><td>78049-100</td><td></td></tr><tr><td>POVIDONE-IODINE</td><td>Thermo Fisher Scientific</td><td>395516</td><td></td></tr><tr><td>OPHTHALMIC OINTMENT</td><td>Thermo Fisher Scientific</td><td>NC0490117</td><td></td></tr><tr><td>Styker T/Pump Warm Water Recirculator</td><td>Kent Scientific Corporation</td><td>TP-700</td><td></td></tr><tr><td>1ml Sub-Q Syringe</td><td>Thermo Fisher Scientific</td><td>309597</td><td></td></tr><tr><td>ENCORE Sensi-Touch PF</td><td>Moore Medical LLC</td><td>30347</td><td>Latex, powder-free surgical glove</td></tr><tr><td>PrecisionGlide 25G Hypodermic Needles</td><td>Thermo Fisher Scientific</td><td>14-826-49</td><td></td></tr><tr><td>Ultra-High-Temperature Tungsten Wire,</td><td>McMaster-Carr</td><td>3775K37</td><td>0.005&quot; Diameter, 1/16 lb. Spool, 380&#039; Long</td></tr><tr><td>304 stainless steel, 24G thin walled tubing</td><td>Microgroup Inc</td><td>304h24tw-5ft</td><td></td></tr><tr><td>#15 Scalpel Blades</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>#10 Scalpel Blades</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>Narrow Pattern Forceps</td><td>Fine Science Tools</td><td>11002-12</td><td>Serrated/Straight/12cm</td></tr><tr><td>Iris Forceps</td><td>Fine Science Tools</td><td>11066-07</td><td>1x2 Teeth/Straight/7cm</td></tr><tr><td>Dissector Scissors</td><td>Fine Science Tools</td><td>14081-09</td><td>Slim Blades/Angled to Side/Sharp-Sharp/10cm</td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14058-11</td><td>ToughCut/Straight/Sharp-Sharp/11.5cm</td></tr><tr><td>Olsen-Hegar Needle Holder with Suture Cutter</td><td>Fine Science Tools</td><td>12002-12</td><td>Straight/Serrated/12cm/with Lock</td></tr><tr><td>Crile Hemostat</td><td>Fine Science Tools</td><td>13004-14</td><td>Serrated/Straight/14cm</td></tr><tr><td>Tungsten Wire Cutter</td><td>ACE Surgical Supply Co., Inc.</td><td>08-051-90</td><td>ACE #150 Wire Cutter, tungsten carbide tips</td></tr><tr><td>3-0 VICRYL Suture</td><td>Ethicon Suture</td><td>J423H</td><td>3-0 VICRYL UNDYED 27&quot; FS-2 CUTTING</td></tr><tr><td>piXarray 100 Digital Specimen Radiography System</td><td>Bioptics, Inc</td><td></td><td>Cabinet x-ray system</td></tr><tr><td>Einhorn 3-Point Bending Device</td><td>N/A</td><td>N/A</td><td>Custom Built</td></tr></tbody></table>

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 successful implementation of the surgical procedure was monitored with radiographic imaging. Key steps include the insertion of an intramedullary needle, the placement of a guide wire, the induction of a transverse fracture at the femoral midshaft, and the proper stabilization with an intramedullary rod (Figure 2Ai - 2Aiv). The healing progression of the fracture callus was monitored with weekly radiographic images up to ...

Watch this Scientific Journal Video about The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing at JoVE.com

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone 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-generation-closed-femoral-fractures-mice-model-to-study-bone

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Medicine

Unit 1

Bone Tissue and the Skeletal System

SCIENTISTS IN ACTION

15.1K Views.  Indiana University School of Medicine. 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.

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

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; ...

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 ...

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 ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

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. ...

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 ...

Biology

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.

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 ...