We thank the staff and veterinarians at the Scott &#38; White Hospital Department of Comparative Medicine, Temple, Texas, for their invaluable advice and assistance during the development of this technique. This work was funded in part by The Institute for Regenerative Medicine Program Funds, Scott &#38; White RGP grant #90172, NIH 2P40RR017447-07 and NIH R01AR066033-01 (NIAMS). We thank Dr Suzanne Zeitouni for proofing the manuscript.

NOTE: This protocol is designed for female nude (Nu/J) mice (18-25 g, 6 weeks) acquired from Jackson Laboratories. Since strains of mice vary slightly in terms of anatomy and growth rate, we advise that the fabrication of pins is optimized to the strain, gender and age of the recipients prior to implantation into live subjects. If the strains are carefully matched, the interference fit between the pin and marrow cavity is highly reproducible. Procedures for housing, diet and general animal husbandry are beyond the scope ...

<ol>
	<li>Brinker, M. R., O'Connor, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+of+fractures+and+dislocations+referred+for+orthopaedic+services+in+a+capitated+population.">The incidence of fractures and dislocations referred for orthopaedic services in a capitated population.</a> J Bone Joint Surg Am. 86, 290-297 (2004).</li><li>Cheung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+bone+healing.">The future of bone healing.</a> Clin Podiatr Med Surg. 22, 631-641 (2005).</li><li>Rosemont, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> United States Bone and Joint Decade: The burden of musculoskeletal diseases and musculoskeletal injuries. , (2008).</li><li>Tzioupis, C., 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=Prevalence+of+long-bone+non-unions.">Prevalence of long-bone non-unions.</a> Injury. 38, S3-S9 (2007).</li><li>Marsh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concepts+of+fracture+union,+delayed+union,+and+nonunion.">Concepts of fracture union, delayed union, and nonunion.</a> Clin Orthop Relat Res. , S22-S30 (1998).</li><li>Spicer, P. 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=Evaluation+of+bone+regeneration+using+the+rat+critical+size+calvarial+defect.">Evaluation of bone regeneration using the rat critical size calvarial defect.</a> Nat Protoc. 7, 1918-1929 (2012).</li><li>Green, E., Lubahn, J. D., Evans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors,+treatment,+and+outcomes+associated+with+nonunion+of+the+midshaft+humerus+fracture.">Risk factors, treatment, and outcomes associated with nonunion of the midshaft humerus fracture.</a> J Surg Orthop Adv. 14, 64-72 (2005).</li><li>Kanakaris, N. K., Giannoudis, P. 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The authors have no competing financial interests.

Herein, we describe a simple method to generate a critical-sized pin-stabilized defect of the murine femur using standard laboratory and veterinary equipment. While the assembly of the pins and the surgical procedure itself requires practice, it is well within the capabilities of a well-trained biomedical research scientist or veterinarian.
The pin is positioned into the medullary canal without additional fixation, making the procedure technically more feasible than more complicated approaches...

It is estimated that half of the US population experience a fracture by the age of 651. For those patients with fractures treated surgically, 500,000 procedures involve the use of a bone graft2 and this number is expected to rise with an increasingly aging population3. Although bone is one of the few organs that has the capacity to completely heal without scarring, there are instances where the process fails3,4. Depending on the circumstances and quality of treatment, 2-30% of long bone fractures fail, resulting in non-union3,5. While there remains some debate on the definition, pseudoarthrosis, critical-sized or non-union bone injuries generally refers to an injury that does not heal over the natural lifetime of the subject6. For experimental purposes, this duration is shortened to the average time required for complete healing of an average-sized bone injury. Non-union bone lesions occur for numerous reasons, but major factors include extreme trauma resulting in a critically-sized gap, infection, poor angiogenesis, tobacco use, or inhibited osteoregenerative capacity due to disease or age7. Even if non-unions are successfully treated, it can cost in excess of $60,000 per procedure, depending on the type of injury and the approaches employed8.
In moderate cases, autologous bone grafting is employed. This strategy involves recovery of bone from a donor site and implantation at the site of injury. While this approach is extremely effective, the volume of available donor-derived bone is limited and the procedure involves an additional surgery, which results in persistent pain in many patients9,10. In addition, the efficacy of the autologous bone graft is dependent of the health of the patient. Bone substitutes made from synthetic materials or processed cadaveric bone are abundantly available11-13, but they have significant limitations, including poor host-cell adhesion properties, reduced osteoconductivity, and the potential for immune rejection14. There is therefore an urgent need for bone regeneration technologies that are safe, effective and widely available.
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. Although it can vary with species15, this is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section16. 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.
For human cell-based applications, immune-compromised mice are attractive to work with because they are physiologically well-characterized, easy to house, cost effective, and easily analyzed radiologically and histologically. Of paramount importance is that immune-compromised mice do not reject cells from different species including humans. Their small size also permits the testing of very small numbers of cells or volumes of experimental scaffolds in orthopedic applications. Several murine orthopedic models have been reported that afford various degrees of bone stability17,18. Those systems that result in very high levels of stability, such as external fixators and locking plates predominantly heal by intramembranous ossification although endochondral healing has been reported19. In contrast, those that permit some micro- and/or macro-motion, such as those employing unfixed or partially-fixed medullary pins, generally heal with a predominance of endochondral ossification20,21. Delayed union or non-union defects of long bone are particularly difficult to achieve in mice due to the extra level of stabilization required. However, a number of approaches have been reported, including medullary pins with interlocking nails, locking plates and external fixators22. These systems generally work well, but given their complicated design they can be technically challenging to install. For example, Garcia et al.23 devised an elegant interlocking pin system for use in mice, but the procedure involves incisions at two separate sites and extensive modification of the femur to accommodate the pins. These procedures were performed under a dissecting microscope.
Herein, we describe a simple femoral medullary pin with a central collar designed to prevent closing of a 3 mm bone deficit and also delineate the original edges of the defect. While the pin was not fixed to the bone itself, precise sizing of the pin diameter and reaming of the medullary cavity results in sufficient interference to minimize torsional motion (Figure 1). With careful selection of inbred age, gender and strain-matched mice, the result is a highly reproducible hypertrophic non-uniondefect22 which can be easily evaluated radiologically. Moreover regions of interest can be reproducibly defined after micro-computed tomography (&#181;CT) for measurement of de novo bone formation and histomorphological parameters. The pins were prototyped in our laboratory using readily available tools.
<img alt="Figure 1" src="/files/ftp_upload/52368/52368fig1highres.jpg" /> 
Figure 1: Experimental principle. Diagrammatic summary of the segmental defect model. The central 3 mm segment of a 9-10 mm murine femur is excised surgically (left). A 3 mm long, 19 gauge surgical steel tube is passed over a 9 mm long, 22 G stainless steel tube and fixed with adhesive at the exact center (right). The resultant pin is fitted into the medullary canals of the remaining proximal and distal portions of the femur with the 19 G collar replacing the 3 mm segment of bone (below, center).

<table border="0" cellpadding="0" cellspacing="0" width="799">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">Name of Equipment/Material*</td>
			<td style="width:207px;">Company</td>
			<td style="width:180px;">Catalog or model</td>
			<td style="width:179px;">Notes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pin Assembly</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dremel rotary tool</td>
			<td>Dremel</td>
			<td>8220</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heavy duty cut off wheel</td>
			<td>Dremel</td>
			<td>420</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tubing 19G</td>
			<td>Small Parts (Amazon)</td>
			<td>B000FMZ8LY</td>
			<td>OD 1.07mm, ID 0.889mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tubing 21G</td>
			<td>Small Parts (Amazon)</td>
			<td>B000FMZ8YQ</td>
			<td>OD 0.82mm, ID 0.635mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tubing 22G</td>
			<td>Small Parts (Amazon)</td>
			<td>B000FMYLZS</td>
			<td>OD 0.719mm, ID 0.502mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tubing 23G</td>
			<td>Small Parts (Amazon)</td>
			<td>B000FN0SY0</td>
			<td>OD 0.643mm, ID 0.444mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cyanoacrylate adhesive</td>
			<td>Loctite</td>
			<td>1365882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Emery disc</td>
			<td>Dremel</td>
			<td>413</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rubber polishing point</td>
			<td>Dremel</td>
			<td>462</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Felt polishing disc</td>
			<td>Dremel</td>
			<td>414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin sponge</td>
			<td>Surgifoam/Ethicon</td>
			<td>1974</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Punch biopsy cutter</td>
			<td>Miltex</td>
			<td>33-34</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgery/post-operative</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Warm pad and circulator pump</td>
			<td>Stryker/Thermocare</td>
			<td>TP700, TP700C, TPP722</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coverage quaternary spray</td>
			<td>Steris</td>
			<td>1429-77</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bead sterilizer</td>
			<td>Germinator/CellPoint Scentific</td>
			<td>Germinator 500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia system</td>
			<td>VetEquip Inc</td>
			<td>901806 or 901807/901809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isofluorane anesthetic</td>
			<td>VETone/MWI</td>
			<td>501017, 502017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical disinfectant</td>
			<td>Chloraprep/CareFusion</td>
			<td>260449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tools</td>
			<td>Fine Science Tools</td>
			<td>various</td>
			<td>recommend German made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Face protection</td>
			<td>Splash Shield</td>
			<td>4505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rechargable high speed drill</td>
			<td>Fine Science Tools</td>
			<td>18000-17</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diamond cutting wheel</td>
			<td>Strauss Diaiond</td>
			<td>361.514.080HP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Absorbable sutures&#160;</td>
			<td>Covidien</td>
			<td>UM-213</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Outer sutures</td>
			<td>Ethicon</td>
			<td>668G</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vetbond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydration gel</td>
			<td>Clear H2O</td>
			<td>70-01-1082</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diet gel</td>
			<td>Clear H2O</td>
			<td>72-01-1062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenorphine</td>
			<td>Reckitt and Benckser</td>
			<td>12496-0757-01</td>
			<td>controlled substance</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse igloos</td>
			<td>Bio Serv</td>
			<td>K3328, 3570,3327</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Euthanasia cocktail</td>
			<td>Euthasol/Virbac</td>
			<td>710101</td>
			<td>controlled substance</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live animal imager&#160;</td>
			<td>Orthoscan</td>
			<td>FD Pulse</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-CT unit and software</td>
			<td>Bruker</td>
			<td>Skyscan1174</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sealing film/Parafilm M</td>
			<td>VWR or Fisher</td>
			<td>100501-338, S37441</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">*Generic sources are suitable for all other items such as gause, drapes, protective clothing, animal care equipment.</td>
		</tr>
	</tbody>
</table>

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.

Mice typically recover consciousness and hind-limb mobility 5-10 minutes after withdrawal of anesthesia. During the first 5 days, it is advisable to house mice individually and introduce environmental enrichment to prevent excess use of the limb. For this purpose, igloo-type nests reduce the need for nest building and encourage resting. We have also observed that provision of food and hydrogel on the floor of the cage reduces the probability of pin displacement. During the 5-day period, analgesia should be administered a...

Watch this Scientific Journal Video about A Simple Critical-sized Femoral Defect Model in Mice at JoVE.com

A Simple Critical-sized Femoral Defect Model in Mice

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 ...

a-simple-critical-sized-femoral-defect-model-in-mice

Research

JoVE Journal

Medicine

14.9K 조회수.  Texas A&M Health Science Center. 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. 