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) ...

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