Name: An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice (Video) | JoVE Journal
Uploaded: 2016-11-13T15:00:00.000+00:00
Duration: 7 min 41 s
Description: Learn intramedullary locking nail fixation for standardized femur osteotomy in mice. This reproducible surgical technique enables precise analysis of normal and defective bone healing mechanisms in vivo.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by RISystem AG, Davos, Switzerland.

All procedures were IACUC-approved and followed institutional guidelines (Landesamt f&#252;r Verbraucherschutz, Zentralstelle Amtstier&#228;rztlicher Dienst, Saarbr&#252;cken, Germany). Analgesia and infection prevention should be in agreement with the respective guidelines of the country and institution where the experiments are to be performed.
1. Preparation of Implants and Surgical Instruments
<ol>
	<li>Select a scalpel blade (size 15), small preparation scissors, fine forceps, dressing forceps, small pincers, 24 gauge (G) and 27 G needles, a non-resorbable 5-0 suture, and a needle holder from the microsurgical instrument box.</li>
	<li>Unpack the intramedullary nail, the interlocking pins, the special aiming device, the Gigli saw, the template for the Gigli saw, the centering drill bit (1 mm diameter), the drill bit (0.3 mm diameter), and the hand drill (Figure 2; see List of Materials). 
	NOTE: The intramedullary nail (0.8 mm diameter, 15.7 mm length) is an intramedullary locking nail made of medical-grade stainless steel for retrograde implantation into the femur. The nail has a proximal thread (4 mm length) and two holes for the insertion of the interlocking pins (0.3 mm diameter) to achieve axial and rotational stability (Figure 1).</li>
	<li>Expose the implants and all surgical instruments to a disinfecting solution (e.g., 96% alcohol) for 5 min or sterilize them (steam sterilization, 130 &#176;C, 25 min). After disinfection or sterilization, place the instruments on a sterile operation cloth. Position the sterile operation cloth directly adjacent to the small animal operation table.</li>
</ol>
2. Animals, Anesthesia, and Analgesia
<ol>
	<li>Choose the strain, age, and sex of the mice as necessary for the study and the question to be addressed. 
	NOTE: For this study 12- to 14-week-old male CD-1 mice were used. For nail implantation, the ideal body weight of the animals is 25 - 35 g.</li>
	<li>Anesthetize the mice with an intraperitoneal injection of 15 mg/kg xylazine and 75 mg/kg ketamine. Confirm the anesthetization by toe pinch. Apply eye lubricant to protect the animals&#39; eyes from drying during anesthesia. After induction of anesthesia, place the mouse under a heat lamp to keep the body temperature constant.</li>
	<li>Apply tramadol-hydrochloride in the drinking water (2.5 mg/100 ml) for analgesia from day 1 before the surgery until day 3 after the surgery.</li>
</ol>
3. Surgical Procedure and Nail Implantation
<ol>
	<li>Before surgery, shave the entire right hind leg and apply a depilatory cream. After 5 min, remove the cream and clean the leg with water.&#160;Expose the implants and all surgical instruments to a disinfecting solution (e.g., 96% alcohol) or sterilize them (steam sterilization,130 &#176;C, 25 min).</li>
	<li>Under aseptic conditions, place the mouse in the supine position on the small animal operation table. Bend the right knee to allow for an anterior approach to the condyles of the knee. Perform a 5 mm medial parapatellar incision at the right knee using the scalpel blade.</li>
	<li>Lift the patellar ligament with the fine forceps and mobilize the ligament carefully with the scalpel blade. Then, shift the patella laterally with the scalpel blade to expose the intercondylar notch of the femur.</li>
	<li>Open the intercondylar notch by drilling until the intramedullary cavity is reached.
	<ol>
		<li>Start drilling with a 45&#186; offset to the femur axis using the 1 mm centering drill bit. Slowly change the direction of the drill bit during drilling until it parallels the bone axis of the femur. Stop drilling if the intramedullary cavity is reached.</li>
	</ol>
	</li>
	<li>After opening the bone at the intercondylar notch, insert the 24 G needle into the intramedullary cavity over the whole length of the femur. Ream the intramedullary cavity of the femur manually through rotary motions of the 24 G needle. Remove the 24 G needle and insert the thinner 27 G needle into the intramedullary cavity. Push the needle forward to perforate the cortical bone of the femur proximally at the greater trochanter.</li>
	<li>Remove the 27 G needle from the femur. Using the hand drill, implant the intramedullary nail through the intercondylar notch under continuous rotation and axial pressure until the distal end of the nail reaches the level of the condyles. 
	NOTE: The distal end of the nail can be identified with a small mark.</li>
	<li>Place the mouse in the left lateral position. Perform a longitudinal skin incision using the scalpel blade along the diaphyseal part of the lateral femur from the knee joint to the hip joint in order to surgically expose the midshaft of the femur.</li>
	<li>Using small preparation scissors, split the fascia and spread the muscles in the direction of the femur axis from the lateral side. Spread the muscles until the diaphyseal part of the femur is exposed. Preserve the sciatic nerve.
	<ol>
		<li>Prepare the whole circumference of the femur by undermining the bone with the dressing forceps. Then, retract the muscles by spreading the dressing forceps and expose the femur.</li>
	</ol>
	</li>
	<li>Mount the aiming device to the distal end of the nail. Advance the device until it attaches to the adapter flange of the nail and turn the aiming device in anterolateral position to the femur.</li>
	<li>Interlock the nail with a proximal and a distal interlocking pin.
	<ol>
		<li>Start with the proximal interlocking pin.</li>
		<li>Insert the centering drill bit (1 mm diameter) into the hand drill. Countersink the bone at the proximal interlocking hole position. 
		NOTE: By countersinking, a small cavity is created in the facing cortical bone without drilling through the bone. This cavity allows for improved centering and guiding of the thinner drill bit (0.3 mm diameter), used later.</li>
		<li>Insert the drill bit (0.3 mm diameter) into the hand drill. Using the aiming device, drill the hole through both the facing and the averted cortical bone (bicortical). Insert the first interlocking pin through the aiming device. The interlocking pin drive shaft shears off as soon as the interlocking torque is achieved.</li>
		<li>Repeat this procedure for the distal interlocking pin.</li>
	</ol>
	</li>
	<li>Perform the diaphyseal osteotomy.
	<ol>
		<li>Attach the saw guide to the aiming device on the lateral side between the two interlocking pins. Then, saw the bone with the Gigli saw under continuous irrigation with saline. After the osteotomy is completed, cut the saw at one end, close to the bone. Remove the saw carefully to avoid causing damage to the soft tissue.</li>
	</ol>
	</li>
	<li>Remove the aiming device and, with the small pincers, clip off the remaining shaft of the intramedullary nail at the marked line.</li>
	<li>Close the muscle layers at the lateral site of the femur and perform the skin closure with single sutures. At the anterior site of the knee, reposition the patella and fix the patella tendon to the muscles with one single suture. Use single sutures to close this wound as well.</li>
	<li>Keep the animals under the heat lamp until they recover from anesthesia. Do not leave the animals unattended until they have regained sufficient consciousness to maintain ventral recumbency. Return the animals to single cages in the animal facility.&#160;</li>
	<li>Monitor the animals carefully every day. Maintain postoperative analgesia during the first three days. Continue analgesia if, on day 4 after surgery, the animals still show evidence of pain, as indicated by vocalization, restlessness, lack of mobility, failure to groom, abnormal posture, and lack of normal interest in surroundings. Terminate analgesia when the animals are pain free.</li>
</ol>

<ol><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+animal+bone+healing+models%3A+standards%2C+tips%2C+and+pitfalls+results+of+a+consensus+meeting%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting.</a> Bone. 49 (4), 591-599 (2011).</li>
<li>Claes, L., Augat, P., Suger, G., Wilke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+size+and+stability+of+the+osteotomy+gap+on+the+success+of+fracture+healing%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">Influence of size and stability of the osteotomy gap on the success of fracture healing.</a> J Orthop Res. 15 (4), 577-584 (1997).</li>
<li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+healing+process+in+non-stabilized+and+stabilized+femur+fractures+in+mice%5BTitle%5D)+AND+%22Arch+Orthop+Trauma+Surg%22%5BJournal%5D)">Characterization of the healing process in non-stabilized and stabilized femur fractures in mice.</a> Arch Orthop Trauma Surg. 136 (2), 203-211 (2016).</li>
<li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+analysis+of+rotational+stiffness+of+different+osteosynthesis+techniques+in+mouse+femur+fracture%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">Ex vivo analysis of rotational stiffness of different osteosynthesis techniques in mouse femur fracture.</a> J Orthop Res. 27 (9), 1152-1156 (2009).</li>
<li>Holstein, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+stable+closed+femoral+fracture+model+in+mice%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">Development of a stable closed femoral fracture model in mice.</a> J Surg Res. 153 (1), 71-75 (2009).</li>
<li>Garcia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+LockingMouseNail-a+new+implant+for+standardized+stable+osteosynthesis+in+mice%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">The LockingMouseNail-a new implant for standardized stable osteosynthesis in mice.</a> J Surg Res. 169 (2), 220-226 (2011).</li>
<li>Cheung, K. M., Kaluarachi, K., Andrew, G., Lu, W., Chan, D., Cheah, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+externally+fixed+femoral+fracture+model+for+mice%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">An externally fixed femoral fracture model for mice.</a> J Orthop Res. 21 (4), 685-690 (2003).</li>
<li>Garcia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+technique+for+internal+fixation+of+femoral+fractures+in+mice%3A+impact+of+stability+on+fracture+healing%5BTitle%5D)+AND+%22J+Biomech%22%5BJournal%5D)">A new technique for internal fixation of femoral fractures in mice: impact of stability on fracture healing.</a> J Biomech. 41 (8), 1689-1696 (2008).</li>
<li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+internal+locking+plate+to+study+intramembranous+bone+healing+in+a+mouse+femur+fracture+model%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.</a> J Orthop Res. 28 (3), 397-402 (2010).</li>
<li>Thompson, Z., Miclau, T., Hu, D., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+model+for+intramembranous+ossification+during+fracture+healing%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">A model for intramembranous ossification during fracture healing.</a> J Orthop Res. 20 (5), 1091-1098 (2002).</li>
<li>Hiltunen, A., Vuorio, E., Aro, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+standardized+experimental+fracture+in+the+mouse+tibia%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">A standardized experimental fracture in the mouse tibia.</a> J Orthop Res. 11 (2), 305-312 (1993).</li>
<li>Manigrasso, M. B., O'Connor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+closed+femur+fracture+model+in+mice%5BTitle%5D)+AND+%22J+Orthop+Trauma%22%5BJournal%5D)">Characterization of a closed femur fracture model in mice.</a> J Orthop Trauma. 18 (10), 687-695 (2004).</li>
<li>Lovati, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetic+mouse+model+of+orthopaedic+implant-related+Staphylococcus+aureus+infection%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Diabetic mouse model of orthopaedic implant-related Staphylococcus aureus infection.</a> PLoS One. 8 (6), e67628(2013).</li>
<li>Slade Shantz, J. A., Yu, Y. Y., Andres, W., Miclau, T. 3rd, Marcucio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+macrophage+activity+during+fracture+repair+has+differential+effects+in+young+adult+and+elderly+mice%5BTitle%5D)+AND+%22J+Orthop+Trauma%22%5BJournal%5D)">Modulation of macrophage activity during fracture repair has differential effects in young adult and elderly mice.</a> J Orthop Trauma. 28, 10-14 (2014).</li>
<li>Claes, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+mechanical+factors+on+the+fracture+healing+process%5BTitle%5D)+AND+%22Clin+Orthop+Relat+Res%22%5BJournal%5D)">Effects of mechanical factors on the fracture healing process.</a> Clin Orthop Relat Res. 355 (Suppl), 132-147 (1998).</li>
<li>Gröngröft, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fixation+compliance+in+a+mouse+osteotomy+model+induces+two+different+processes+of+bone+healing+but+does+not+lead+to+delayed+union%5BTitle%5D)+AND+%22J+Biomech%22%5BJournal%5D)">Fixation compliance in a mouse osteotomy model induces two different processes of bone healing but does not lead to delayed union.</a> J Biomech. 18 (13), 2089-2096 (2009).</li>
<li>Glatt, V., Matthys, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adjustable+stiffness%2C+external+fixator+for+the+rat+femur+osteotomy+and+segmental+bone+defect+models%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models.</a> J Vis Exp. (9), e51558(2014).</li>
<li>Manassero, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+murine+femoral+segmental+critical-sized+defect+model+stabilized+by+plate+osteosynthesis+for+bone+tissue+engineering+purposes%5BTitle%5D)+AND+%22Tissue+Eng+Part+C+Methods%22%5BJournal%5D)">A novel murine femoral segmental critical-sized defect model stabilized by plate osteosynthesis for bone tissue engineering purposes.</a> Tissue Eng Part C Methods. 19 (4), 271-280 (2013).</li>
</ol>

Romano Matthys is an employee of RISystem AG, Davos, Switzerland, which produces the implants and implant-specific instruments. The other authors have no conflicts of interest.

The most critical steps of the surgical technique are the correct positioning of the nail, the aiming device, and the pins. The nail has to be inserted completely to the marked indent at the distal end of the nail, because a protrusion of the nail into the knee joint at the level of the condyles can restrict the movement of the knee (Figure 3 A). Therefore, the size of the femur and, accordingly, the body weight of the animals, must be considered. The surgeon should also pay special attention to the fina...

The biology of bone healing may be studied in vitro using cell and spheroid cultures, but it also requires in vivo approaches using animal studies. While large-animal experiments still play an important role in preclinical testing, early stage testing of products or hypotheses has changed during the last 10 years and is nowadays often conducted in small animal models1. This switch was performed for several reasons. Production and maintenance of mice and rats are cheaper compared to pigs and sheep. In addition, small animals have shorter reproduction times and shorter normal healing periods, both of which facilitate the performance of large series of chronic experiments. Finally, the availability of gene-targeted animals and specific antibodies allows for the analysis of molecular mechanisms in bone healing. However, while the previously used osteosynthesis techniques in the larger animal models could be translated with minimal variation from similar procedures used in human or veterinary clinical patient care, the development and application of osteosynthesis techniques in the small-sized rats and mice turned out to be challenging.
It is well known that the biomechanical environment significantly influences the bone healing process2. As known from fracture healing in humans, differences in fracture stabilization result in distinct modes of healing, including intramembranous ossification after rigid fixation and endochondral ossification after less rigid fixation with micromovements. Complete axial or rotational instability may delay the healing process or may result in non-unions3. Accordingly, we feel that it is necessary to develop sophisticated implant systems and osteosynthesis techniques in mice and rats. In this way, the biomechanical conditions can be standardized appropriately, guaranteeing valid results when analyzing the healing process.
Although a considerable number of highly sophisticated murine stabilization techniques have been introduced during the last few years, the most commonly used technique is still the simple intramedullary pin. The major disadvantage of this technique, however, is the lack of rotational and axial stability4. To improve rotational and axial stability, an intramedullary screw was introduced to stabilize femur fractures in mice5. However, the screw fixation cannot be used to analyze bone-defective healing due to the need for contact and compression between the bone fragments in order to maintain rotational stability.
The intramedullary locking nail offers higher axial and rotational stability compared to the simple pin and the intramedullary screw4. A highly reproducible femur osteotomy, possible because of the guide for the Gigli saw and the ability to create defined gap sizes, allows for the analysis of both normal bone healing and bone-defective healing6. Due to the insertion of interlocking pins, the intramedullary locking nail guarantees a constant gap size during the entire healing process, even while bearing full weight. Here, we report on the design and application of the intramedullary locking nail, as well as on its advantages and disadvantages in experimental studies on normal and delayed bone healing.

<table><tbody><tr><td>MouseNail</td><td>RISystem AG</td><td>221,122</td><td></td></tr><tr><td>MouseNail aiming device</td><td>RISystem AG</td><td>221,201</td><td></td></tr><tr><td>MouseNail interlocking pin</td><td>RISystem AG</td><td>221,121</td><td></td></tr><tr><td>Centering bit</td><td>RISystem AG</td><td>592,205</td><td></td></tr><tr><td>Drill bit</td><td>RISystem AG</td><td>590,200</td><td></td></tr><tr><td>Gigli wire saw</td><td>RISystem AG</td><td>590,100</td><td></td></tr><tr><td>Suture (5-0 Prolene)</td><td>Ethicon</td><td>8614H</td><td></td></tr><tr><td>Forceps</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>BD520R</td><td></td></tr><tr><td>Dressing forceps</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>BJ009R</td><td></td></tr><tr><td>Scissors</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>BC100R</td><td></td></tr><tr><td>Needle holder</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>BM024R</td><td></td></tr><tr><td>24 G needle</td><td>BD Mircolance 3</td><td>304100</td><td></td></tr><tr><td>27 G needle</td><td>Braun Melsungen AG</td><td>9186182</td><td></td></tr><tr><td>Scalpel blade size 15</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>16600525</td><td></td></tr><tr><td>Pincers</td><td>Knipex</td><td>7932125</td><td></td></tr><tr><td>Heat radiator</td><td>Sanitas</td><td>605.25</td><td></td></tr><tr><td>Depilatory cream</td><td>Asid bonz GmbH</td><td>NDXZ10</td><td></td></tr><tr><td>Eye lubricant</td><td>Bayer Vital GmbH</td><td>2182442</td><td></td></tr><tr><td>Xylazine</td><td>Bayer Vital GmbH</td><td>1320422</td><td></td></tr><tr><td>Ketamine</td><td>Serumwerke Bernburg</td><td>7005294</td><td></td></tr><tr><td>Tramadol</td><td>Gr&amp;uuml;nenthal GmbH</td><td>2256241</td><td></td></tr><tr><td>Disinfection solution (SoftaseptN)</td><td>Braun Melsungen AG</td><td>8505018</td><td></td></tr><tr><td>CD-1 mice</td><td>Charles River</td><td>22</td><td></td></tr></tbody></table>

an intramedullary locking nail for standardized fixation of femur osteotomies to analyze normal and defective bone healing in mice

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.

The overall time for the surgical procedure was about 30 min from skin incision to wound closure. Using the surgical implants provided, surgery can be performed without a stereo-microscope. Postoperatively, the animals were monitored daily. Post-operative analgesia was terminated after 3 days because none of the animals showed evidence of pain (vocalization, restlessness, lack of mobility, failure to groom, abnormal posture, or lack of normal interest in surroundings) after this time peri...

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 ...

The overall goal of this surgical technique is to standardize the analysis of bone healing in mice. This surgical technique can help answer key questions in bone healing research about the roots of bone morphogenetic proteins and muscular grow factors and the mechanisms of bone repair. The main advantages of this technique are the reproducibility of the femur osteotomy and a high axial and rotationist ability facilitated by the use of the intramedullary locking nail.Demonstrating the procedure will be Tobias Fritz, a colleague of our department. One day before the surgery until three days after the surgery, add tramadol hydrochloride to the animal's drinking water for analgesia. On the day of the surgery, confirm the appropriate level of sedation of a 25 to 35 gram mouse, by a lack of response to toe pinch and place the animal under a heat radiator.Shave the entire right hind leg of the animal and apply depilatory cream. After five minutes, remove the cream and clean the leg with water. Then disinfect the surgical site with 96%ethanol.Under aseptic conditions, place the mouse in the supine position on the operating table with the right knee bent to allow an anterior approach of the knee condyles. Next, use a sterilized size 15 scalpel blade to make a five millimeter medial parapatellar incision in the right knee. Then use fine forceps to lift the exposed patellar ligament and use the blade to carefully mobilize the ligament.Shifting the patella laterally with the blade to expose the intercondylar notch of the femur, use a hand drill equipped with a one millimeter centering bit, and held at a 45 degree offset to the femur axis to bore the intercondylar notch. Slowly changing the direction of the bit, drill until the bit parallels the bone axis of the femur, stopping when the intramedullary cavity is reached. When the intercondylar notch is open, insert a 24 gauge needle into the cavity over the entire length of the femur and rotate the needle to manually ring the intramedullary cavity.Then replace the 24 gauge needle with a 27 gauge needle and push the 27 gauge needle forward to perforate the cortical bone of the femur proximally at the greater trochanter. Remove the 27 gauge needle and use the hand drill to implant the intramedullary nail through the intercondylar notch under continuous rotation and axial pressure until the distal end of the nail reaches the condyles. Now place the mouse in the left lateral position and use the scalpel to make a longitudinal skin incision along the diaphyseal region of the lateral femur from the hip joint to the knee joint to surgically expose the midshaft of the femur.Using small preparation scissors, split the fascia and spread the muscles toward the femur axis from the lateral side until the diaphyseal region of the femur is exposed. Undermine the bone with the dressing forceps. Then spread the dressing forceps and retract the muscles to expose the femur.Mount the aiming device to the distal end of the nail, advancing the device until it attaches to the adapter flange of the nail. Then turn the aiming device to an anterolateral angle to the femur, and use the centering one millimeter diameter drill bit to create a small cavity in the facing cortical bone at the proximal interlocking hole position. Next, with a 0.3 millimeter diameter drill bit, use the aiming device to drill a hole through both the facing and averted cortical bones.Insert the proximal interlocking pin through the aiming device. The interlocking pin driveshaft will shear off as soon as the interlocking torque is achieved. When both the proximal and distal interlocking pins have been placed, equip the aiming device with the saw guide on the lateral side, between the two interlocking pins, and saw the bone with a Gigli saw under continuous irrigation with saline.When the osteotomy is complete, cut the saw at end close to the bone and carefully remove the blade to avoid causing damage to the soft tissue. Remove the aiming device and use small pincers to clip off the remaining shaft of the intramedullary nail at the marked line. Then close the muscle layers at the lateral site of the femur, and use single sutures to close the skin.At the anterior site of the knee, reposition the patella and fix the patella tendon to the muscles with one single suture, followed by closure of the wound with single sutures. Finally, allow the animal to recover under the heat lamp with monitoring, until fully recovered. The most significant complication that can occur is the incorrect implantation of the locking nail with the protrusion of the nail level to the condyles of the knee joint.This mainly occurs due to the incorrect handling of the aiming device or the use of an animal with a too small femur, particularly in mice with body weights below 20 grams. Another complication is the dislocation of an interlocking pin, which is mainly caused by an incomplete insertion of the pin, and can be avoided by radiographic confirmation of the correct implant placement during, or immediately after, the surgery. Radiological analysis after five weeks confirms a complete healing of the 0.25 millimeter osteotomy gap, at which point, the periosteal callus is almost completely remodeled.In contrast, in femora stabilized with a 2 millimeter gap, the osteotomy does not heal, and reliably exhibits an atrophic nonunion formation for up to 10 weeks of bone healing. After stabilization with a 0.25 millimeter osteotomy gap, histological analysis reveals a typical pattern of secondary fracture healing with callus formation, including intramembranous and endochondral ossification that is remodeled into lamellar bone at five weeks. In contrast, femora stabilized with two millimeter osteotomy gaps demonstrate an atrophic nonunion after 10 weeks that is associated with a high amount of fibrous tissue within the osteotomy gap, and no evidence of bone healing or bridging.Once mastered, this technique can be completed in 20 minutes if it is performed properly. After watching this video, you should have a good understanding of how to use this technique as the standardest model for fracture healing research.

an-intramedullary-locking-nail-for-standardized-fixation-femur

Research

JoVE Journal

Medicine

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.

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

Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection 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.

A Simple Critical-sized Femoral Defect Model in Mice

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.

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

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

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

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

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.

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

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.

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

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Transverse Fracture of the Mouse Femur with Stabilizing Pin

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Establishing a Diaphyseal Femur Fracture Model in Mice

Here, we present a protocol for accessing the guidewire of intramedullary femoral nailing in obese patients using an in-house designed awl. Using the new awl with a distal positioner for the guidewire insertion and opening the bone can increase efficiency in inserting the guidewire and reduce the difficulty of surgery.

Treatment with Locking Intramedullary Nailing for Intertrochanteric Fracture of the Femur Utilizing a New Awl with a Distal Positioner

Learn intramedullary locking nail fixation for standardized femur osteotomy in mice. This reproducible surgical technique enables precise analysis of normal and defective bone healing mechanisms in vivo.

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice (Video) | JoVE Journal

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

Explore More Videos