Name: an intramedullary locking nail for standardized fixation of femur osteotomies to analyze normal and defective bone healing in mice
Uploaded: 2016-11-13T15:00:00
Description: Watch this Scientific Journal Video about An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice at JoVE.com

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 ...

<ol>
	<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>Claes, L., Augat, P., Suger, G., Wilke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+size+and+stability+of+the+osteotomy+gap+on+the+success+of+fracture+healing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+healing+process+in+non-stabilized+and+stabilized+femur+fractures+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+analysis+of+rotational+stiffness+of+different+osteosynthesis+techniques+in+mouse+femur+fracture.">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. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+externally+fixed+femoral+fracture+model+for+mice.">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/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> J Biomech. 41 (8), 1689-1696 (2008).</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> 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+intramembranous+ossification+during+fracture+healing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+experimental+fracture+in+the+mouse+tibia.">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. 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Y., Andres, W., Miclau, T., Marcucio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+macrophage+activity+during+fracture+repair+has+differential+effects+in+young+adult+and+elderly+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+factors+on+the+fracture+healing+process.">Effects of mechanical factors on the fracture healing process.</a> Clin Orthop Relat Res. 355 (Suppl), 132-147 (1998).</li><li>Gr&#246;ngr&#246;ft, I., 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=Fixation+compliance+in+a+mouse+osteotomy+model+induces+two+different+processes+of+bone+healing+but+does+not+lead+to+delayed+union.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjustable+stiffness,+external+fixator+for+the+rat+femur+osteotomy+and+segmental+bone+defect+models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+murine+femoral+segmental+critical-sized+defect+model+stabilized+by+plate+osteosynthesis+for+bone+tissue+engineering+purposes.">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>

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 border="0" cellpadding="0" cellspacing="0" width="476">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:187px;">MouseNail</td>
			<td style="width:179px;">RISystem AG</td>
			<td style="width:111px;">221,122</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MouseNail aiming device</td>
			<td>RISystem AG</td>
			<td>221,201</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MouseNail interlocking pin</td>
			<td>RISystem AG</td>
			<td>221,121</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Centering bit</td>
			<td>RISystem AG</td>
			<td>592,205</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Drill bit</td>
			<td>RISystem AG</td>
			<td>590,200</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gigli wire saw</td>
			<td>RISystem AG</td>
			<td>590,100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture (5-0 Prolene)</td>
			<td>Ethicon</td>
			<td>8614H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Forceps</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BD520R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dressing forceps</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BJ009R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissors</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BC100R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle holder</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BM024R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">24G needle</td>
			<td>BD Mircolance 3</td>
			<td>304100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">27G needle</td>
			<td>Braun Melsungen AG</td>
			<td>9186182</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scalpel blade size 15</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>16600525</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pincers</td>
			<td>Knipex</td>
			<td>7932125</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat radiator</td>
			<td>Sanitas</td>
			<td>605.25</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Depilatory cream</td>
			<td>Asid bonz GmbH</td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NDXZ10</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Eye lubricant</td>
			<td>Bayer Vital GmbH</td>
			<td>2182442</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazine</td>
			<td>Bayer Vital GmbH</td>
			<td align="right">1320422</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ketamine</td>
			<td>Serumwerke Bernburg</td>
			<td align="right">7005294</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tramadol</td>
			<td>Gr&#252;nenthal GmbH</td>
			<td align="right">2256241</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Disinfection solution (SoftaseptN)</td>
			<td>Braun Melsungen AG</td>
			<td align="right">8505018</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CD-1 mice</td>
			<td>Charles River</td>
			<td align="right">22</td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice at JoVE.com

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

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.

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