Name: an intramedullary locking nail for standardized fixation of femur osteotomies to analyze normal and defective bone healing in mice
Uploaded: 2016-11-13T15:00:00.000+00:00
Duration: 7 min 41 s
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 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/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. 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=Development+of+a+stable+closed+femoral+fracture+model+in+mice.">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/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> 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/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. P. <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+a+closed+femur+fracture+model+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+mouse+model+of+orthopaedic+implant-related+Staphylococcus+aureus+infection.">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., 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>

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

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 ...

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13.3K Views.  Saarland University. 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.

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

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 ...

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

Developmental Biology

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 ...

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 ...

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

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

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

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.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

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

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

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.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

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 ...

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 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 ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

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

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

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

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