Name: an efficient and reproducible protocol for distraction osteogenesis in a rat model leading to a functional regenerated femur
Uploaded: 2017-10-23T15:00:00
Description: Watch this Scientific Journal Video about An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur at JoVE.com

This work was supported and funded by the CNRS Mecabio challenge.
The authors thank the animal care technician for taking care of the animals throughout the procedure. The authors also acknowledge IVTV central Lyon, through Thierry Hoc. Thanks to Marjorie Sweetko for language revision.
We are greatful to Maryl&#232;ne Lallemand, C&#233;cile G&#233;nov&#233;sio, and Patrick Laurent for their contribution to this experimental study.

All procedures described were approved by the University of Aix-Marseille institutional animal care and use committee and the French research ministry and performed in the conventional animal house of Marseille Medical Faculty (France).
1. Define Functional Specifications of the External Fixator Based on the Following Guidelines
<ol>
	<li>Optimize bone anchorage.
	<ol>
		<li>Implant half-threaded pins with a diameter (of the threaded section) of 1 mm.</li>
	</ol>
	</li>
	<li...

<ol>
	<li>Jauregui, J. J., Ventimiglia, A. V., Grieco, P. W., Frumberg, D. B., Herznberg, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerate+Bone+stimulation+following+limb+lengthening:+a+meta-analysis.">Regenerate Bone stimulation following limb lengthening: a meta-analysis.</a> BMC Musculoskelet Disord. 17 (1), 407 (2016).</li><li>Morcos, M. W., Al-Jallad, H., Hamdy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+review+of+Adipose+Stem+Cells+and+Their+Implication+in+Distraction+Osteogenesis+and+Bone+Regeneration.">Comprehensive review of Adipose Stem Cells and Their Implication in Distraction Osteogenesis and Bone Regeneration.</a> Biomed Res Int. 2015 (842975), 1-20 (2015).</li><li>Cans&#252;, E., &#220;nal, M. B., Parmaksizoglu, F., G&#252;rcan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distraction+lengthening+of+the+proximal+phalanx+in+distal+thumb+amputations.">Distraction lengthening of the proximal phalanx in distal thumb amputations.</a> Acta Orthop Traumatol Turc. 49 (3), 227-232 (2014).</li><li>Singh, M., Vashistha, A., Chaudhary, M., Kaur, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Basis+of+Distraction+Osteogenesis+-+A+Review.">Biological Basis of Distraction Osteogenesis - A Review.</a> J Oral Maxillofac Surg Med Pathol. 28 (1), 1-7 (2016).</li><li>Sailhan, F., Gleyzolle, B., Parot, R., Guerini, H., Viguier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rh-BMP-2+in+Distraction+Osteogenesis:+Dose+Effect+and+Premature+Consolidation.">Rh-BMP-2 in Distraction Osteogenesis: Dose Effect and Premature Consolidation.</a> Injury. 41 (7), 680-686 (2010).</li><li>Schiller, J. R., Moore, D. C., Ehrlich, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Lengthening+Rate+Decreases+Expression+of+Fibroblast+Growth+Factor+2,+Platelet-Derived+Growth+Factor,+Vascular+Endothelial+Growth+Factor,+and+CD31+in+a+Rat+Model+of+Distraction+Osteogenesis.">Increased Lengthening Rate Decreases Expression of Fibroblast Growth Factor 2, Platelet-Derived Growth Factor, Vascular Endothelial Growth Factor, and CD31 in a Rat Model of Distraction Osteogenesis.</a> J Pediatr Orthop. 27 (8), 961-968 (2007).</li><li>Aronson, J., Shen, X. C., Skinner, R. A., Hogue, W. R., Badger, T. M., Lumpkin, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Model+of+Distraction+Osteogenesis.">Rat Model of Distraction Osteogenesis.</a> J Orthop Res. 15 (2), 221-226 (1997).</li><li>Aida, T., Yoshioka, I., Tominaga, K., Fukuda, J. <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+latency+period+in+a+rabbit+mandibular+distraction+osteogenesis.">Effects of latency period in a rabbit mandibular distraction osteogenesis.</a> Int J Oral Maxillofac Surg. 32, 54-62 (2003).</li><li>Lammens, J., Liu, Z., Aerssend, J., Dequeker, J., Fabry, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=distraction+Bone+Healing+Versus+Osteotomy+Healing:+A+Comparative+Biochemical+Analysis.">distraction Bone Healing Versus Osteotomy Healing: A Comparative Biochemical Analysis.</a> J Bone Miner Res. 13 (2), 279-286 (1998).</li><li>Isefuku, S., Joyner, C. J., Simpson, A. H. R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Murine+Model+of+Distraction+Osteogenesis.">A Murine Model of Distraction Osteogenesis.</a> Bone. 27 (5), 661-665 (2000).</li><li>Eberson, C. P., Hogan, K. A., Moore, D. C., Ehrlich, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Low-Intensity+Ultrasound+Stimulation+on+Consolidation+of+the+Regenerate+Zone+in+a+Rat+Model+of+Distraction+Osteogenesis.">Effect of Low-Intensity Ultrasound Stimulation on Consolidation of the Regenerate Zone in a Rat Model of Distraction Osteogenesis.</a> J Pediatr Orthop. 23 (1), 46-51 (2003).</li><li>&#214;zdel, A., Saris&#246;zen, B., Yal&#231;inkaya, U., Demirag, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+HIF+Stabilizer+on+Distraction+Osteogenesis.">The Effect of HIF Stabilizer on Distraction Osteogenesis.</a> Acta Orthop Traumatol Turc. 49 (1), 80-84 (2015).</li><li>Takamine, Y., 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=Distraction+Osteogenesis+Enhanced+by+Osteoblastlike+Cells+and+Collagen+Gel.">Distraction Osteogenesis Enhanced by Osteoblastlike Cells and Collagen Gel.</a> Clin Orthop Relat Res. 399, 240-246 (2002).</li><li>Wang, X., Zhu, S., Jiang, X., Li, Y., Song, D., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+Administration+of+Lithium+Improves+Distracted+Bone+Regeneration+in+Rats.">Systemic Administration of Lithium Improves Distracted Bone Regeneration in Rats.</a> Calcif Tissue Int. 96 (6), 534-540 (2015).</li><li>Xu, J., 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=Human+Fetal+Mesenchymal+Stem+Cell+Secretome+Enhances+Bone+Consolidation+in+Distraction+Osteogenesis.">Human Fetal Mesenchymal Stem Cell Secretome Enhances Bone Consolidation in Distraction Osteogenesis.</a> Stem Cell Res Ther. 7 (1), (2016).</li><li>Yasui, N., 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=Three+Modes+of+Ossification+during+Distraction+Osteogenesis+in+the+Rat.">Three Modes of Ossification during Distraction Osteogenesis in the Rat.</a> Bone Joint J. 79 (5), 824-830 (1997).</li><li>Chang, F., 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=Stimulation+of+EP4+Receptor+Enhanced+Bone+Consolidation+during+Distraction+Osteogenesis.">Stimulation of EP4 Receptor Enhanced Bone Consolidation during Distraction Osteogenesis.</a> J Orthop Res. 25 (2), 221-229 (2007).</li><li>Nyman, J. S., 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=Quantitative+Measures+of+Femoral+Fracture+Repair+in+Rats+Derived+by+Micro-Computed+Tomography.">Quantitative Measures of Femoral Fracture Repair in Rats Derived by Micro-Computed Tomography.</a> J Biomech. 42 (7), 891-897 (2009).</li><li>Hsu, J. T., Wang, S. P., Huang, H. L., Chen, Y. J., Wu, J., Tsai, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assessment+of+trabecular+bone+parameters+and+cortical+bone+strength:+a+comparison+of+micro-CT+and+dental+cone-beam+CT.">The assessment of trabecular bone parameters and cortical bone strength: a comparison of micro-CT and dental cone-beam CT.</a> J Biomech. 46, 2611-2618 (2013).</li><li>Xue, J., 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=NELL1+Promotes+High-Quality+Bone+Regeneration+in+Rat+Femoral+Distraction+Osteogenesis+Model.">NELL1 Promotes High-Quality Bone Regeneration in Rat Femoral Distraction Osteogenesis Model.</a> Bone. 48 (3), 485-495 (2011).</li><li>Mark, H., Bergholm, J., Nilsson, A., Rydevik, B., Str&#246;mberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+External+Fixation+Method+and+Device+to+Study+Fracture+Healing+in+Rats.">An External Fixation Method and Device to Study Fracture Healing in Rats.</a> Acta Orthop. 74 (4), 476-482 (2003).</li></ol>

This study describes a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator. Our DO protocol led to a functional regenerated bone. After 47 days of consolidation, removal of the homemade external fixator and 2 days of physiological weight-bearing by the animal did not induce any fracture of the regenerating callus. Thanks to the micro-CT reconstructi...

Distraction osteogenesis (DO) is a surgical technique widely used clinically1,2,3,4 in humans for lower1,2 and upper3 limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision as well as in maxillofacial reconstruction4. DO leads to bone regeneration after placement of an external fixator in bone and osteotomy. The osteotomized extremities are moved away from each other by gradual distraction2 to reach the desired elongation. A consolidation period follows, during which there is no more elongation.
The DO procedure is divided into three distinct phases: latency, distraction, and consolidation. Generally, a 7-day latency period starts just after osteotomy4. This allows bone repair to begin the initial step of the healing process4. The latency period is followed by a distraction period where traction forces are applied to the regenerated callus and surrounding soft tissues1,2,4. When the desired elongation is reached, distraction stops and the consolidation period begins. During this period, the external fixator is maintained until the regenerated bone is functional enough to support its removal.
Various parameters of DO influence bone repair such as length and rate of lengthening, type of external fixator, frequency of distraction, length of the consolidation period, or type of mechanical stress applied to the distracted callus. As an example, the rate and frequency of lengthening can lead either to premature consolidation5 or disruption of the process by creating non-recoverable damage like necrotic tissue or cysts within the callus6,7.
Many DO protocols have been applied to different animal models8,9,10 to study bone repair processes and to maximize bone consolidation. In rats, most studies11,12,13,14,15 focused on how to shorten the DO protocol by speeding up callus consolidation. Some of these experimental studies used external fixators already commercially available for human clinical applications5,13,15,16. However, these types of external fixator are not suitable for DO on the rat femur, which exhibits different anatomical characteristics from the human femur. Moreover, only a few studies clearly demonstrate the efficiency of their protocols in obtaining a functional regenerated bone7,16. It is therefore difficult to compare results from various DO studies, due to their differing protocols and lack of information regarding the external fixator12,13,14,17.
Thus, the aim of this study was to describe, in a rat model, an efficient and reproducible protocol for DO on the femur that leads to a functional regenerated bone. To this end, we designed a homemade and easy-to-use external fixator especially for the rat femur, which we have described in detail in this protocol. In drafting the technical specifications for this device, we took into account all the fundamental constraints for a good distribution of mechanical stresses and avoiding the production of residual stress. The technical specification included an appropriate geometry for the device to allow pure traction force on bones and surrounding tissue, an appropriate weight for the gait of the animal, control of the length of bone elongation, and a good alignment of bone segments without production of shear stress at the intersection of pins and bone. Moreover, this device had to be usable without sedation of the animal during distraction, biocompatible, and sterilizable without damage. After 7 weeks of consolidation, this protocol for DO on the rat femur led to a functional regenerated bone, demonstrated by the animals&#39; physiological weight-bearing without fracture of the regenerated callus after removal of the external fixator. The physiological gait of the animals was consistent with architectural parameters obtained from micro-CT analysis of regenerated callus and X-ray analysis.

<table>
	<tbody>
		<tr>
			<td>K&#233;tamine</td>
			<td>Renaudin</td>
			<td>578 540-2</td>
			<td>Supply by animal house</td>
		</tr>
		<tr>
			<td>M&#233;d&#233;tomidine</td>
			<td>Virbac</td>
			<td>6799091</td>
			<td>Supply by animal house</td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Centravet</td>
			<td>567 477-2</td>
			<td>Supply by animal house</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Indivor France</td>
			<td>3400932731060</td>
			<td>Supply by animal house</td>
		</tr>
		<tr>
			<td>Enrofloxacine</td>
			<td>ChannelPharmaceutical Facturing</td>
			<td>FR/V/4955220</td>
			<td>Supply by animal house</td>
		</tr>
		<tr>
			<td>Piezotome</td>
			<td>Satelec Acteon</td>
			<td>F57510</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pet pad</td>
			<td>Therasage</td>
			<td>AL8365936</td>
			<td>Supply by the animal house</td>
		</tr>
		<tr>
			<td>Dental X-ray</td>
			<td>S.A.R.L Innovation m&#233;dicales et dentaires</td>
			<td>WYZ - BLUEX</td>
			<td></td>
		</tr>
		<tr>
			<td>Winiwix Software</td>
			<td>Softys Dental</td>
			<td>PFT</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT system</td>
			<td>nanoScan SPECT/CT</td>
			<td>GEIT-31105EN (05/14)</td>
			<td>Subcontract by IVTV central Lyon</td>
		</tr>
		<tr>
			<td>Micro-CT analysis Software</td>
			<td>phoenix datos X2 reconstruction</td>
			<td>none</td>
			<td>Free software</td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td>Brawn</td>
			<td>GT415</td>
			<td>Supply by animal house</td>
		</tr>
		<tr>
			<td>Senn&#8217;s retractors</td>
			<td>Word Precision Instruments</td>
			<td>501718</td>
			<td>Blunt version</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Mundipharma Medical Company</td>
			<td>D08AG02</td>
			<td>Supply by animal house</td>
		</tr>
		<tr>
			<td>Resorbable suture thread (5.0)</td>
			<td>Ethicon</td>
			<td>JV1023</td>
			<td>Supply by animal house</td>
		</tr>
		<tr>
			<td>Rugine</td>
			<td>Word Precision Instruments</td>
			<td>503406</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo-Hegar needle holder</td>
			<td>Word Precision Instruments</td>
			<td>V503382</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal drill</td>
			<td>Beuterlock</td>
			<td>V020944018003</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Olsen-Hegar Needle-holder</td>
			<td>Word Precision Instruments</td>
			<td>501989</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo scissor</td>
			<td>Word Precision Instruments</td>
			<td>501752</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Word Precision Instruments</td>
			<td>500236</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley</td>
			<td>Janvier</td>
			<td>none</td>
			<td>12 weaks and male</td>
		</tr>
	</tbody>
</table>

an efficient and reproducible protocol for distraction osteogenesis in a rat model leading to a functional regenerated femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

The X-ray images taken from the end of surgical procedure to the end of consolidation showed no loosening of the half-threaded pins in the femur, indicating stable anchorage. The pins were parallel and well-preserved. The osteotomized extremities were well-aligned along the longitudinal axis of the bone during the DO process (Figure 2). At the end of the latency period, no calcified areas were visible (Figure 2B)...

Watch this Scientific Journal Video about An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur at JoVE.com

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

The overall goal of this protocol is to reproduibly and efficiently induce femoral distraction osteogenesis in a rat model to assess functional bone regeneration. This method can help answer key questions in the osteogenesis medicine field about determining and shortening the consolidation period or about identifying the mechanism of bone repair. The main advantage of this technique is vital to who made external fixator and to guide who is doing the surgery, provide a stable environment for optimal bone regeneration.The implications of this technique extend to all therapeutic upper and lower limb lengthening or of bone defect that occur after bone tumor excision surgery. The demonstration of the procedure is done by Edouard Lamy, a pharmacy lecturer from my laboratory. Begin by placing an anesthetized 12-week old male Sprague Dawley rat in the left lateral position and removing the hair from the right hind limb.After applying eye ointment, disinfect the surgical area with Povidone-iodine solution and place the animal in the supine position. With the external fixator positioned along the mediolateral axis, use a marker to make one point at the knee and a second point at the hip along the medial line of the sagittal plane. Connect the two points and use a scalpel to make a skin incision along the line, cutting between the biceps femoris and the vastus lateralis until the femur is fully exposed.Lift the periosteum and use a rougine to disconnect the soft tissue from the bone. Insert pins into the most proximal and distal holes of the external fixator. If both pins can be anchored, use two Senn's retractors to open the muscles and tighten the clamp of a drilling guide in the middle of the femur, starting with the proximal and distal holes, and finishing with the middle two holes, pass a metal 0.6 millimeter drill bit through the bone to create four guide holes.Using a 0.8-millimeter half-threaded pin, work the pin back and forth to enlarge the four pre-holes, taking care to stay perpendicular to the femur. To implant the pins, use a needle holder to grasp the head of each 1-millimeter half-thread pin and individually sink the pins to enlarge the guide holes. Use a Senn's retractor to confirm that the pins penetrate both of the cortices without protruding more than 1 millimeter.When the pins are in place, connect the external fixator to the half-threaded pins, making sure that the offset is about 6 millimeters to allow easy stitching and good rigidity. Then secure the four locking screws so that the external fixator is locked to the pins. Using a Piezotome, perform an osteotomy between the two central pins.Then use a Mayo-Hegar Needle Holder, a resorbable 5-0 suture thread and a continuous stitch to close the wound. Immediately after the surgery, x-ray the animal to check the depth of the pins and the alignment of the osteotomized extremities along the long axis. Then allow rat to recover fully with monitoring.After one week, x-ray the limb to recheck the positioning of the pins and the alignment of the bone segments. To perform the distraction, manually turn the square nut one-half turn clockwise every 12 hours for 10 days. X-ray the limb halfway through and at the end of the distraction process to monitor the positioning of the pins and the alignment of the bone segments.At the end of the distraction, x-ray the limb weekly, removing the external fixator on day 64. On day 66, use a scalpel to make a skin incision through the surgery scar from the top of the hip to the front of the knee. Cut between the biceps femoris and the vastus lateralis until the femur is fully exposed, disconnecting the muscle from the bone as much as possible.Cut all of the ligaments of the knee, dismantle the articulation, and cut the joint capsule of the hip. Clean the bone thoroughly without removing the pins, using a scalpel to remove all of the soft tissue. When the contralateral femur has been cleaned, x-ray both femurs.Remove the pins and store the femurs at minus 20-degree Celsius for micro-computed tomography scan analysis. In this representative experiment, x-ray imaging from immediately after the surgery through the end of the consolidation, revealed that the half-threaded pins within the femur pins remained parallel, well preserved, and unloosened throughout the procedure, indicative of a stable anchorage. Moreover, the osteotomized extremities had clearly been well aligned along the longitudinal axis of the bone during the distraction osteogenesis process.At the end of the latency period, no calcified areas were visible near the osteotomized extremities. At the end of the distraction period, a few calcified areas were visible close to the pre-existing cortices. After 28 days of consolidation, the non-calcified region of the gap between the osteotomized extremities was smaller, and a periosteal callus could be observed near the gap level with the pins.After 47 days of consolidation, the regenerating callus was completely bridged. After removal of the external fixator and two days of physiological weightbearing, the animals displayed a physiological gait and no evidence of fracture could be observed. 3D micro-computed tomography analysis of the serial longitudinal sections of the regenerating callus demonstrated that calcified bridging was always present with a continuous outer cortical observed at the periphery of the regenerating callus.After 49 days of consolidation, no fracture was observed despite the persistence of a less mineralized region at the center of the regenerated callus. Once mastered, this surgical technique can be completed in 45 minutes if it is performed properly. While attempting this procedure, it is important to remember to maintain a sterile environment during the surgery and to thoroughly monitor the animal after the surgery.After it's development, this technique pave the way for researcher in the field of medicine to improve the distraction osteogenesis technique, particularly for shortening the consolidation period in the clinical practice. Following this procedure, other methods like adding growth factors or build materials can be performed to answer additional questions such as, can the consolidation of the bone be improved. After watching this video, you should have a good understanding of how to perform femoral lengthening in a rat model using these techniques as a guide to ensure parallelism between the pins.

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Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...