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/...

<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. 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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. 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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. 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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>
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			<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&#34; Diameter, 1/16 lb. Spool, 380&#39; 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>
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		<tr>
			<td>Iris Forceps</td>
			<td>Fine Science Tools</td>
			<td>11066-07</td>
			<td>1x2 Teeth/Straight/7cm</td>
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			<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&#34; 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 ...

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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 ...

This method can help answer key questions in fracture healing research about the biological mechanisms of repair and the development of novel therapeutic strategies for promoting efficient bone healing. The main advantage of this technique is that it serves as minimally invasive procedure for generating standardized fractures to study bone fracture healing in mice. Demonstrating the surgical procedure will be Yong Li and Anuradha Valiya Kambrath, technicians from our laboratory.After confirming the appropriate level of sedation by toe pinch, apply ointment to the eyes of an anesthetized 10-week old male mouse and remove the fur from the right hind limb. Starting at the center of the knee and making a circular sweep outward, disinfect the exposed skin with three sequential iodine-based and 70%ethanol scrubs, and place the mouse on a heating pad covered by a sterile surgical drape in the supine position. Flex the knee of the operative leg and use a scalpel blade to make a 1.5 centimeter incision centered over the knee joint.Use forceps to laterally displace the patella, exposing the distal end of the femur and insert a 1.5 inch long, 25 gauge stainless steel hypodermic needle at the center of the trochlear groove in a retrograde direction down the length of the medullary canal and through the proximal end of the femur. Obtain an X-ray to ensure the proper placement of the pin. Then, pass a four-inch long, 36 gauge tungsten guidewire through the shaft of the needle into the hub at the distal femur and exiting the bevel on the dorsal side of the mouse.Following a successful placement of the guidewire, gently pull the hub to carefully remove the needle while holding the limb and guidewire in place, and confirm the placement of the guidewire by X-ray. Now, place the mouse in the gravity dependent three-point bending device, positioning the femur horizontally across the two supporting points, such that the intertrochanteric and supracondylar regions of the femur rest on the support anvils and the lateral side of the limb is facing the loading point. The most critical step of this protocol is placement of the femur within the three-point bending apparatus, as the fracture geometry is dependent on the applied bending force and the positioning of the hind limb.When the femur is in position, position a 391 gram weight, 34.6 centimeters above the impact disc of the device and drop the weight. Immediately, but carefully, remove the mouse from the device and confirm the fracture location by X-ray. Insert a piece of 24 gauge stainless steel hypodermic tubing over the guidewire to stabilize the fractured bone, and confirm the position of the steel rod and the stabilization of the fractured femur by X-ray.Remove the guidewire and use wire cutters to remove any excess tubing at the distal end of the femur. Using forceps, apply a gentle downward force to bury the exposed tubing under the surface of the condyles, taking care not to dislocate the knee joint, and reposition the patella. Then, close the incision site with a 5-0 absorbable suture.Once the surgeries have been completed, inject each mouse with up to 500 microliters of sterile saline, intraperitoneally, to aid in their postoperative recovery, and monitor the animals until their full recumbency. Weekly monitoring of the healing progression of the fracture callus up to 28 days following the surgery, reveals a prominent soft callus formed from mineralized cartilage by day 14 post-fracture, which is remodeled into a hard callus by day 21 post-fracture. Toluidine blue staining reveals the formation of a cartilage matrix at the fracture gap by day seven, that becomes aligned with the fracture gap by day 14, post-fracture.Staining for Type one collagen expression illustrates the spatial organization and relative amounts of bone matrix that is present 14 days post-fracture. Micro-computed tomography analysis demonstrates a decrease in the callus volume of approximately 50%between days 14 and 28, post-fracture, indicating an effective remodeling of the callus. Before attempting this procedure, it is important to determine the weight and drop height, as they are dependent on the age, sex, and strain of the mice.Radiographic imaging is commonly used to monitor the healing progression, while techniques such as immunohistochemistry and micro-CT can be used to answer questions about the mechanisms of bone regeneration and to assess the extent of the bone fracture healing. The development of this technique has provided a platform for the study of complex biological mechanisms of fracture healing and for exploring novel therapeutic strategies that accelerate bone tissue regeneration in mice.

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14.9K vistas.  Indiana University School of Medicine. This method can help answer key questions in fracture healing research about the biological mechanisms of repair and the development of novel therapeutic strategies for promoting efficient bone healing. The main advantage of this technique is that it serves as minimally invasive procedure for generating standardized fractures to study bone fracture healing in mice. Demonstrating the surgical procedure will be Yong Li and Anuradha Valiya Kambrath, technicians from our laboratory.After c...