Name: a minimally invasive model to analyze endochondral fracture healing in mice under standardized biomechanical conditions
Uploaded: 2018-03-22T15:00:00
Description: Watch this Scientific Journal Video about A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions at JoVE.com

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

All procedures were performed according to the National Institutes of Health guidelines for the use of experimental animals and followed institutional guidelines (Landesamt f&#252;r Verbraucherschutz, Zentralstelle Amtstier&#228;rztlicher Dienst, Saarbr&#252;cken, Germany).
1. Preparation of Surgical Instruments and Implants
<ol>
	<li>Select a scalpel blade (size 15), a small swab, fine forceps, a 27 G needle, a non-resorbable 5-0 suture, scissors and a needle holder from the microsurgical i...

<ol>
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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>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>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>Histing, T., Klein, M., Stieger, A., Stenger, D., Steck, R., Matthys, R., Holstein, J. H., Garcia, P., Pohlemann, T., Menger, M. D. <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+model+to+analyze+metaphyseal+bone+healing+in+mice.">A new model to analyze metaphyseal bone healing in mice.</a> J Surg Res. 178 (2), 715-721 (2012).</li><li>Histing, T., Menger, M. D., Pohlemann, T., Matthys, R., Fritz, T., Garcia, P., Klein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.</a> J Vis Exp. (117), (2016).</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>Holstein, J. H., Menger, M. D., Culemann, U., Meier, C., Pohlemann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+locking+femur+nail+for+mice.">Development of a locking femur nail for mice.</a> J Biomech. 40 (1), 215-219 (2007).</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, S132-S147 (1998).</li></ol>

Critical steps of the surgical procedure are to find the correct entry point for screw implantation in the middle of the femur condyles at the intercondylar notch as well as the optimal orientation of the needle parallel to the bone axis for reaming of the intramedullary cavity. To avoid an incorrect entry position, the surgeon should prepare the notch until an optimal view is achieved. To control the orientation during reaming, the femur of the mice should be held with the fingers in a stable position. A further critica...

Bone healing studies in mice are in great demand because of a broad spectrum of antibodies and genetically-modified animals. These facts allow to study the molecular mechanisms of bone healing1. In the past few years, different bone healing models for mice have been developed2. These models can be divided into open models, in which the bone is osteotomized using an open lateral surgical approach and in closed models, in which the bone is fractured based on the fracture model introduced by Bonnares and Einhorn3. Using this technique, a standardized transverse fracture can be produced by a 3-point bending device and intramedullary implants can be inserted through a small medial parapatellar incision in a minimally invasive technique avoiding a major soft tissue trauma.
The intramedullary screw can be applied for closed fracture stabilization in mice. The screw offers rotational and axial stability. This is achieved by fracture compression through a proximal thread and a distal head4. Further advantages of the screw are the simple surgical technique, the low grade of invasivity, the low implant weight and, most notably, a higher stability providing standardized and controlled biomechanical conditions compared to other intramedullary implants5. In fact, in the most closed fracture models, the fragments are stabilized only by simple pins, which is associated with a complete lack of rotational and axial stability and a high risk of pin and also fracture dislocation. This can markedly influence the healing process, which may result in delayed healing or non-union formation.
It is well known that the stability of the fracture fixation has a tremendous impact on the healing process6,7. A high rigid fixation results in intramembranous healing, while a less rigid fixation, which may allow micromovements in the fracture gap, results in endochondral healing. Stabilization of the fracture with the intramedullary screw shows predominantly an endochondral healing with a large amount of callus tissue, particularly after 2 weeks of fracture healing. The possibility to harvest a large amount of callus tissue enables the analysis of multiple parameters by different techniques.
Here, we report on the design and application of the intramedullary screw in mice, as well as on its advantages and disadvantages in experimental studies on normal endochondral bone healing.

<table>
	<tbody>
		<tr>
			<td>Mouse Screw</td>
			<td>RISystem AG</td>
			<td>221,100</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide wire</td>
			<td>RISystem AG</td>
			<td>521,100</td>
			<td></td>
		</tr>
		<tr>
			<td>Centering bit</td>
			<td>RISystem AG</td>
			<td>590,205</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand drill</td>
			<td>RISystem AG</td>
			<td>390,130</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton-Swab (150 mm, small head)</td>
			<td>Fink Walter GmbH</td>
			<td>8822428</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 &#38;CoKG</td>
			<td>BD520R</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Braun Aesculap AG &#38;CoKG</td>
			<td>BC100R</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Braun Aesculap AG &#38;CoKG</td>
			<td>BM024R</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 &#38;CoKG</td>
			<td>16600525</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&#252;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>
		<tr>
			<td>X-ray Device</td>
			<td>Faxitron MX-20, Faxitron X-ray Corporation</td>
			<td>2321A0988</td>
			<td></td>
		</tr>
		<tr>
			<td>Fracture device small</td>
			<td>RISystem AG</td>
			<td>891,100</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The operating time from skin incision to wound closure was 20 min. The surgery can be performed without a stereo-microscope. Postoperatively, the animals were monitored daily. Post-operative analgesia was terminated after 2 days because none of the animals showed evidence of pain after this time period. The animals showed also normal weight-bearing within 2 days after surgery. Wound infections were not observed during the entire observation period.
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Watch this Scientific Journal Video about A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions at JoVE.com

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under 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 overall goal of this surgical technique is to provide standardized conditions for the analysis of fracture healing in mice. This method can help answer key questions in fracture healing research, such as the role of different growth factors in the mechanisms of bone repair. The main advantage of this procedure is that it can be used in a minimally invasive technique and provide standardized biomechanical conditions for the study of endochondral fracture healing in mice.In the following, Philipp, a student, will demonstrate the surgical procedure. Begin by ensuring that all required sterile surgical instruments and materials are positioned on an operation cloth directly adjacent to the small animal operation table. For this study, 12 to 14-week-old male CD-1 mice were used.After inducing anesthesia using an approved method, confirm anesthetization by the lack of response to a toe pinch. Apply eye lubricant to protect the eyes from drying during anesthesia, and place the anesthetized mouse under a heat radiator to keep the body temperature constant. Before surgery shave the entire right hind leg and apply a depilatory cream.After five minutes remove the cream and clean the leg with water. Then apply a disinfecting solution with 96%alcohol. Place the mouse in the supine position on the small animal operating table.Under aseptic conditions, bend the right knee to allow for an anterior approach to the condyles of the knee. Use a scalpel blade to perform a five millimeter medial parapatellar incision at the right knee. Mobilize the patellar ligament carefully with the scalpel blade and the swab.Then use fine forceps to shift the patella laterally to expose the intercondylar notch of the femur. Open the intercondylar notch exactly in the middle of the femur between both condyles. Use a hand drill with a 0.5 millimeter centering drill bit, and start drilling at a slow speed and a 45 degree offset ventrally to the femur axis.During drilling, continuously decrease the angle to zero degrees offset until it is parallel with the bone axis of the femur. Stop drilling when a depth of one millimeter is reached. Insert a 27 gauge needle into the intramedullary cavity over the whole length of the femur and ream the intramedullary cavity of the femur manually using a rotary motion.Then push the needle forward to perforate the cortical bone at the greater trochanter proximally. Remove the 27 gauge needle and apply the guide wire through the distal part of the femur. Then use a size 15 scalpel blade to make a skin incision proximally over the guide wire.Keep the guide wire in place while pushing it forward until both ends of the guide wire are outside. To create a defined closed fracture using the guillotine, place the mouse in a lateral position with the right leg under the guillotine. Make sure that the diaphyseal part of the femur is placed in the middle of the guillotine.Drop the 200 gram weight from the defined distance of 7.6 centimeters. Use the X-ray device to control the fracture configuration and position as well as the position of the guide wire. Use the nose at the distal end of the intramedullary screw to connect it to the 0.2 millimeter guide wire.Insert it into the femur under continuous pressure and clockwise rotation. Shear off the drive shaft when the sufficient torque is achieved, and then remove the guide wire proximally. Reposition the patella, and fix the patella tendon to the muscles with one single suture using a 5-0 synthetic monofilament nonabsorbable polypropylene suture.Use single sutures of the same material and size to close the wound. Use the X-ray device to control the reduction of the fragments and the screw position radiologically. Keep the post-surgical animal under the heat radiator until recovered from anesthesia.Observe the mouse until it has regained sufficient consciousness to maintain ventral recumbency. Return all postoperative animals to single cages in the animal facility. Monitor the animals carefully every day, and provide postoperative analgesia using approved methods for the first three days after surgery.Radiological analyses after two weeks showed an obvious formation of callus tissue bridging the fracture gap. After five weeks the fracture was healed and the periosteal callus was almost completely remodeled. Histological analyses of the callus and the fracture zone after two weeks showed typical distribution of endochondral healing with carticle tissue built during the chondrogenic process and woven bone.After five weeks the cartilage tissue disappeared and the woven bone was converted to the lamellar bone to reconstitute the normal anatomical and load-carrying properties of the bone. Biomechanical analyses after two weeks indicated a bending stiffness of 37%compared to the contralateral unfractured bone. After five weeks the bending stiffness was almost 100%indicating complete healing.Once mastered, this technique can be done in 20 minutes if it's performed properly. After watching this video, you should have a good understanding of how to use this technique as a standardized model for fracture healing research.

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