Name: the generation of closed femoral fractures in mice: a model to study bone healing
Uploaded: 2018-08-16T14:30:00
Description: Watch this Scientific Journal Video about The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing at JoVE.com

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. 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 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&#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>
		</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&#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 ...

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 ...

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.

the-generation-closed-femoral-fractures-mice-model-to-study-bone

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Medicine

Unit 1

Bone Tissue and the Skeletal System

SCIENTISTS IN ACTION

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

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 ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

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 ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

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 ...