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>Choose a design to reduce discomfort for the animal.
	<ol>
		<li>Choose an external fixator of a small size that fits into a volume of 7,723 mm3 (32 mm x 19 mm x 12.7 mm).</li>
		<li>Choose an external fixator with low weight, less than 13 g without the pins, to avoid disturbing the animals&#39; gait. Choose aluminum as the material for the two blocks due to its low density. 
		NOTE: The chamfers of the blocks considerably decrease the weight of the two parts.</li>
	</ol>
	</li>
	<li>Control the motion of the fixator such that the direction of tensile force is maintained parallel to the direction of the bone and ensure pure tensile forces.
	<ol>
		<li>Use a sliding bar that goes through the two blocks . Groove and open the blocks so each can bear two pins.</li>
		<li>Use an elongation screw with one smooth side that turns freely in the block and can be gripped by fingers and with a threaded side that displaces the second block by rotation. Anchor two pins to the bone, on each side of the fracture, to keep the direction of tensile force parallel to the direction of the bone. Maintain the tensile forces in the longitudinal axis to allow good distribution of stress on the pins. Secure the four pins with locking screws.</li>
	</ol>
	</li>
	<li>Choose materials like aluminum, titanium, and steel that can withstand sterilization temperatures.</li>
	<li>Ensure that pin placement corresponds to external fixator geometry (Figure 1-B).
	<ol>
		<li>Use a drilling guide that contains a clamp tightened on the bone to maintain the drilling position.</li>
		<li>Shorten the lever arm of the clamp for low clamping force. Position the clamp in the middle of the device, above the future fracture, to clear the sides.</li>
	</ol>
	</li>
	<li>Make the distraction easy to manual adjust by adding a knurled screw to tighten and release the mobile block.
	<ol>
		<li>Use a screw wide enough to manipulate with fingers alone.</li>
		<li>Add a drilled square nut in the middle of the elongation screw between the two blocks, to allow easy distraction.</li>
		<li>Make this square nut (8 mm on each side) larger than the two blocks to allow manual use with a thin pin or any kind of object. Turn the screw &#188; turn to allow a 0.125 mm lengthening.</li>
	</ol>
	</li>
</ol>
2. Surgery
NOTE: An assistant is required for all surgical procedures. Four 12 week-old male Sprague Dawley rats were fed a standard laboratory diet ad libitum.
<ol>
	<li>Prepare surgical tools.
	<ol>
		<li>Sterilize all the following surgical instruments: 1 rugine, 2 Senn&#39;s retractors, 1 micro Olsen-Hegar Needle-holder, 1 Mayo-Hegar needle holder, 1 mayo scissor, and 1 scalpel.</li>
		<li>Sterilize the external fixator, the drilling guide, 4 half-threaded pins, 4 screws, the tip and the string of the drill, and the tip and the string of the piezotome. Sterilize these instruments by autoclaving at 135 &#176;C for 18 min.</li>
		<li>Install a heating pad below the sterile field, on the surgical table. Place all instruments or tools on another sterile field.</li>
	</ol>
	</li>
	<li>Anesthetize and prepare the animal.
	<ol>
		<li>Weigh the rat to prepare the anesthetic and the analgesic mixture</li>
		<li>Determine and calculate the amount of Bupremorphine and Carprofen to prepare the analgesic mixture extemporaneously. Use Buprenorphine (0.03 mg/mL) and Carprofen (5 mg/mL) respectively at 0.05 mg/kg and 5 mg/kg.</li>
		<li>Restrain the rat and subcutaneously inject the analgesic mixture. &#160;Wait a few seconds, then inject intraperitoneally the anesthetic mixture.</li>
		<li>Shave the right hind limb with an electric razor and disinfect the limb with povidone-iodine solution.</li>
		<li>Lay the animal laterally (right side upward) on the sterile field to allow the correct positioning of the external fixator along the mediolateral axis.</li>
		<li>Put a sterile compress on its head to protect its eyes during surgery.</li>
	</ol>
	</li>
	<li>Implant the external fixator in the femur.
	<ol>
		<li>Landmark the skin incision. With a marker, draw a point from the distal part (knee) and a second point in the proximal part (hip) following the median line in the sagittal plane. Then draw a line between these 2 points.</li>
		<li>Incise the stretched skin along the drawing line using a scalpel.</li>
		<li>Cut in between the biceps femoris and vastus lateralis until the femur is fully exposed, with the help of a scalpel. Use the 2 Senn&#39;s retractors (may require an assistant) to facilitate the muscle incision.</li>
		<li>Lift the periosteum and disconnect the soft tissue from the bone with the help of a rugine.</li>
		<li>Check that the exposed femur is long enough.
		<ol>
			<li>Insert pins in the most proximal and distal holes of the external fixator.</li>
			<li>Position the external fixator and check that both pins can be anchored into the femur.</li>
		</ol>
		</li>
		<li>Implant 4 parallel 1 mm half-threaded pins in the femur.
		<ol>
			<li>Take the drilling guide and move apart the muscles with 2 Senn&#39;s retractors. Tighten the clamp of the drilling guide in the middle of the femur.</li>
			<li>Drill 4 pre-holes in the femur. Using an electric drill, drill holes at a speed of 2,000 rpm by passing a metal drill bit of 0.6 mm diameter through the 4 guide holes.
			<ol>
				<li>Start with the most proximal and distal holes and finish with the two middle ones of the guide. Be careful to go through both cortices but not to damage the soft tissues just beneath the femur.</li>
			</ol>
			</li>
			<li>Take off the drilling guide.</li>
			<li>Enlarge the 4 pre-holes with 0.8 mm half-threaded pin.
			<ol>
				<li>Work the pin back and forth through the 0.6 mm holes. Be careful to stay perpendicular to the femur and sink through both cortices.</li>
			</ol>
			</li>
			<li>Implant 1 mm half-threaded pins.
			<ol>
				<li>Grip the head of the 1 mm half-threaded pin with a needle-holder.</li>
				<li>Sink the pin to enlarge the pre-hole. Check that the pins penetrate both cortices and do not protrude more than 1 mm, with a Senn&#39;s retractor.</li>
			</ol>
			</li>
			<li>Connect the external fixator to the 4 half-threaded pins. Make sure that the offset (distance between the fixator&#39;s two blocks and the bone surface) is about 6 mm to allow easy stitches and good rigidity of the system18.</li>
			<li>Secure the 4 locking screws so that the external fixator is locked to the pins.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Osteotomize the femur.
	<ol>
		<li>Perform an osteotomy between the 2 central pins with a piezotome.</li>
		<li>Close the wound using a continuous stitch with a resorbable suture thread (5.0) and Mayo-Hegar needle holder. Make sure only the skin is stitched, and not the muscles.</li>
	</ol>
	</li>
	<li>Verify the surgery and monitor animals.
	<ol>
		<li>Perform an X-ray just after surgery, while the animal is still anesthetized. Check the depth of all the pins and that osteotomized extremities are aligned following the long axis.</li>
		<li>Ensure analgesia and antibioprophylaxy by subcutaneous injection of Buprenorphine (0.03 mg/mL) and Enrofloxacine (50 mg/mL) respectively at 0.05 mg/kg and 10 mg/kg.&#160;</li>
		<li>Determine and calculate the amount of antipamezole to reverse the anesthesia. Use antipamezole at 1mg/kg and do a subcutaneous injection to administer the product. &#160;Allow the animal to regain consciousness and return it to its cage.</li>
		<li>Six hours after the surgery, inject a second dose of the analgesia mixture (Buprenorphine and Carprofen) respectively at 0.05mg/kg and 5 mg/kg subcutaneously.&#160; 
		For the next 3 days at least, give these injections twice a day for the analgesia mixture and once a day for the antibioprophylaxy. Regular clinical examinations must be performed to assess the effectiveness of analgesia and must be modified according to the behavior signs of each animal. &#160;</li>
		<li>Check that the animals can walk using their operated hind limb normally on the day following surgery.</li>
	</ol>
	</li>
</ol>
3. Distraction
<ol>
	<li>Leave the external fixator in neutral fixation for 1 week. X-ray the limb at the end of this phase. Check the positioning of the pins and the alignment of the bone segments.</li>
	<li>Manually turn the square nut one half turn (0.25 mm) clockwise every 12 h to perform the distraction. Distract for 10 days, which should lead to a 5mm bone lengthening, representing a 12% lengthening relative to the initial length of the operated bone segment.
	<ol>
		<li>Do not anesthetize the animal during this step. X-ray the limb half-way through and at the end of this period to check the positioning of the pins and alignment of the bone segments.</li>
	</ol>
	</li>
	<li>Maintain the external fixator for 47 days. X-ray the limb weekly to check the progress of distracted gap calcification. On Day 64, remove the external fixator and let the animal walk freely for 2 more days (Day 66). The total consolidation period lasts 7 weeks.</li>
	<li>Euthanize all rats after 7 weeks of consolidation with an overdose of inhaled sevoflurane.</li>
	<li>Resect distracted and contralateral femurs without the surrounding tissues.
	<ol>
		<li>Make a skin incision on the scar located on the median line following the sagittal plane with the help of a scalpel. Run the incision from the top of the hip to the front of the knee.</li>
		<li>Cut in between the biceps femoris and vastus lateralis with a scalpel until the femur is fully exposed. As much as possible, disconnect the muscle attached to the bone.</li>
		<li>Cut all the ligaments of the knee and dismantle the articulation.</li>
		<li>Cut the joint capsule of the hip.</li>
		<li>Clean the bone thoroughly without removing the half-threaded pins. Remove all the soft tissue with a scalpel.</li>
	</ol>
	</li>
	<li>Repeat the steps from 3.5.1 to 3.5.5 for the contralateral femur.</li>
	<li>X-ray distracted and contralateral femurs. Remove the 4 half-threaded pins and store all femurs at -20 &#176;C for micro-CT scan analysis (10 &#181;m resolution).</li>
</ol>

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<li>Morcos, M. W., Al-Jallad, H., Hamdy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+review+of+Adipose+Stem+Cells+and+Their+Implication+in+Distraction+Osteogenesis+and+Bone+Regeneration%5BTitle%5D)+AND+%22Biomed+Res+Int%22%5BJournal%5D)">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>
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<li>Schiller, J. R., Moore, D. C., Ehrlich, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+Lengthening+Rate+Decreases+Expression+of+Fibroblast+Growth+Factor+2%2C+Platelet-Derived+Growth+Factor%2C+Vascular+Endothelial+Growth+Factor%2C+and+CD31+in+a+Rat+Model+of+Distraction+Osteogenesis%5BTitle%5D)+AND+%22J+Pediatr+Orthop%22%5BJournal%5D)">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>
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<li>Aida, T., Yoshioka, I., Tominaga, K., Fukuda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+latency+period+in+a+rabbit+mandibular+distraction+osteogenesis%5BTitle%5D)+AND+%22Int+J+Oral+Maxillofac+Surg%22%5BJournal%5D)">Effects of latency period in a rabbit mandibular distraction osteogenesis.</a> Int J Oral Maxillofac Surg. 32, 54-62 (2003).</li>
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<li>Takamine, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distraction+Osteogenesis+Enhanced+by+Osteoblastlike+Cells+and+Collagen+Gel%5BTitle%5D)+AND+%22Clin+Orthop+Relat+Res%22%5BJournal%5D)">Distraction Osteogenesis Enhanced by Osteoblastlike Cells and Collagen Gel.</a> Clin Orthop Relat Res. 399, 240-246 (2002).</li>
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<li>Yasui, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three+Modes+of+Ossification+during+Distraction+Osteogenesis+in+the+Rat%5BTitle%5D)+AND+%22Bone+Joint+J%22%5BJournal%5D)">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/pubmed?term=((Stimulation+of+EP4+Receptor+Enhanced+Bone+Consolidation+during+Distraction+Osteogenesis%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">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/pubmed?term=((Quantitative+Measures+of+Femoral+Fracture+Repair+in+Rats+Derived+by+Micro-Computed+Tomography%5BTitle%5D)+AND+%22J+Biomech%22%5BJournal%5D)">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/pubmed?term=((The+assessment+of+trabecular+bone+parameters+and+cortical+bone+strength%3A+a+comparison+of+micro-CT+and+dental+cone-beam+CT%5BTitle%5D)+AND+%22J+Biomech%22%5BJournal%5D)">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>
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</ol>

The authors have nothing to disclose.

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 reconstruction, the evidence of a complete bridging confirmed that the regenerated callus was functional. A previous study clearly demonstrated a positive correlation between the presence of calcified bridging and the load that a regenerated callus can sustain19. Moreover, the vBMD found for the regenerated callus was about 67% of the vBMD found for the diaphysis part of the femur from the controlateral limb20. We also found an average vBMD of the regenerated callus that was close to the value previously described21. This highlights the stable environment created by the homemade external fixator leading to efficient bone repair. In addition, the fixator was well tolerated by the animals. The positioning and the light weight of the external fixator did not disturb the animals' gaits, so they were able to walk just after surgery.
Throughout the protocol, no breaking or loosening of pins occurred, evidence that the threading procedure ensured an effective and stable anchoring of the pins in the femur. It is crucial that pins are well-anchored for good stability of the DO micro-environment: loosening one pin has been shown to decrease by half the stiffness of the device18. Moreover, rats must be housed singly in cages with a specially designed flat cover to prevent the animal from hooking up the external fixator. The manual and easy-to-perform distraction of the regenerating callus limits pin-loosening but also avoids having to sedate animals every 12 h for several days and contributes to the reliability and efficiency of the homemade external fixator. Another important point to check is that the pins penetrate both cortices and do not protrude more than 1 mm. This step is sensitive, and if X-ray radiographs do not confirm appropriate positioning of the pins, this needs to be corrected manually using a Mayo-Hegar needle-holder while the animal is still anesthetized. The protocol has some limitations. It is a time-consuming and constraining protocol, which limits the number of animals it can be used on. Moreover, given the dexterity required and the meticulousness of the surgery, the procedure could take a couple of practice runs to master.
Throughout the DO procedure, the alignment of the osteotomized extremities was maintained. When implanting pins in the femur, it is very important to use a drilling guide so that the half-threaded pins are parallel, thereby ensuring good distribution of the mechanical stress without production of shear stress throughout the distraction and consolidation phases. It is also very important to clean the wound daily to prevent any blocking of the square nut that would make manual distraction difficult, which could affect the alignment of osteotomized extremities and the proper anchoring of pins. How to proceed to the osteotomy represents the last key point for the regeneration. The osteotomy must be performed with a piezotome to ensure a uniform and regular osseous section and to prevent thermic-induced necrosis and damage to surrounding soft tissues. Moreover, excessive heat is sometimes generated when drilling the pre-holes. An irrigating system coupled with the electric drill can be used to prevent thermic-induced necrosis. 
To conclude, we describe an efficient and reproducible protocol, in a rat femoral model, for DO that leads to a functional regenerated bone. Designing a homemade external fixator based on the anatomical characteristics of the rat femur enabled a stable environment to be generated and allowed the formation of a calcified regenerated callus that was sufficiently consolidated to support physiological weight-bearing by the animals. Our next objective is to use this reproducible protocol to determine the shortest consolidation period compatible with a functional regenerated bone. The subsequent aim will be to improve the DO technique, especially seeking ways to shorten the consolidation period. This reproducible protocol could also be useful to identify mechanisms involved in bone repair. Finally, the characteristics of the home-made external fixator make it usable in clinical practice for human finger elongation.

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&amp;eacute;tamine</td><td>Renaudin</td><td>578 540-2</td><td>Supply by animal house</td></tr><tr><td>M&amp;eacute;d&amp;eacute;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&amp;eacute;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&amp;rsquo;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). At the end of the distraction period, a few calcified areas were visible close to the preexisting cortices (Figure 2C). After 28 days of consolidation (Day 45), the non-calcified region of the gap between the osteotomized extremities was smaller and we observed a periosteal callus not only near the gap but also at the level of the pins (Figure 2D). After 47 days of consolidation (Day 64), the regenerating callus was completely bridged (Figure 2E). After removal of the external fixator and 2 days of physiological weight-bearing, the animals had physiological gait and there was no evidence of fracture (Figure 2F).
3D micro-CT analysis of the serial longitudinal sections of the regenerating callus demonstrated that calcified bridging was always present (Figure 3A-D). A continuous outer cortical was observed at the periphery of the regenerating callus (Figure 3C-D). According to the longitudinal profiles, the regenerating callus was completely filled by a network of bony trabeculae. After 49 days of consolidation (Day 66), a less mineralized region remained at the center of the regenerated bone (Figure 3E-F). The architectural parameters of the micro-CT indicated that the average volume fraction (BV/TV) for the regenerating callus and the contralateral femur were respectively 55% &#177; 13 and 97.85% &#177; 1.7 (Table 1 and Table 2). The mean value of volumetric bone mineral density (vBMD) for the regenerating callus was 750 mg/cm3 &#177; 25. The mean value of the cross-sectional area was higher for the regenerating callus than for the contralateral femur (17.23 mm2 &#177; 9.3 vs 9.5 mm2&#160;&#177; 1.2) (Table 1 and Table 2). The mean callus cortical thickness was thinner than the cortical thickness of contralateral femur (0.317 mm &#177; 0.04 vs 0.6 mm &#177; 0.05) (Tables 1 and 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56433/56433fig1.jpg" /> 
Figure 1: Computer-aided design (CAD) of the external fixator and the drilling guide. (A) CAD of the external fixator with the pins. (B) CAD of the drilling guide. Scale bar = 5 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56433/56433fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56433/56433fig2.jpg" /> 
Figure 2: Representative X-rays of a rat femur at various time-points. X-ray radiographs of the distracted callus at 0, 7, 17, 45, 64 and 66 days. (A) X-rays taken after the surgery (Day 0). (B) X-ray radiograph taken after the 7-day latency period. No calcified callus is apparent near the osteotomized extremities (OE). (C) At the end of distraction, a small, poorly calcified zone is apparent near the osteotomized extremities (rectangular zone). (D) After 28 days of consolidation (Day 45), the gap between the osteotomized extremities (*) and a second cortical is apparent (yellow arrow). (E) Radiograph after 47 days of consolidation (Day 64) (F) X-ray radiograph taken after 49 days of consolidation (Day 66), with two days of physiological weight-bearing. Scale bar = 1 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56433/56433fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56433/56433fig3.jpg" /> 
Figure 3: 3D micro-CT reconstruction of the distracted callus of a rat femur. The mineralization of the bone is illustrated by color ranging from yellow to blue. (A,B) Representation of the bridging cortices of the distracted callus following the anteroposterior and longitudinal axis. (C,D) Proximal and distal cross-section revealing the initial cortical bone (black arrow) and the bridging cortices of the distracted callus (yellow arrow) (E,F) 3D longitudinal profiles of the distracted callus after 7 weeks of consolidation: the callus is filled by a network of trabecular bone. Scale bar = 1000 &#181;m. Calibration bar = 0 to 2.54 g/cm3 (yellow = 1.73 g/cm3, pink = 0.84 g/cm3, and blue = 0.17 g/cm3). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56433/56433fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/56433/56433table1.jpg" /> 
Table 1: Callus parameters of distracted femur after 49 days of consolidation. The region of interest (ROI) selected to calculate all the parameters was the zone between the osteotomized extremities. All the values obtained for each rat were calculated on each slice of the image stack. Results are expressed as mean &#177; standard deviation. BV/TV: volume fraction; CSA: cross-sectional area; vBMD: bone mineral density; Ct.Th: cortical thickness.
<img alt="Table 2" src="/files/ftp_upload/56433/56433table2.jpg" /> 
Table 2: Contralateral parameters of intact femur after 49 days of consolidation. All the values obtained for each rat were calculated on each slice of the image stack. Results are expressed as mean &#177; standard deviation. BV/TV: volume fraction; CSA: cross-sectional area; Ct.Th: cortical thickness.

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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Universidad Científica del Sur

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Bioengineering

7.5K Views.  Aix Marseille Univ. 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.

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

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 ...

Medicine

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

A Mouse Distraction Osteogenesis Model

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

Developmental Biology

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

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. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

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

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 ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials and bone tissue. The rat's tibia is frequently chosen due to its easy surgical access through thin tissue layers (skin and muscle) and the flattened shape of its medial face, facilitating the surgical insertion of intraosseous devices. Additionally, this model enables the induction of specific diseases, mimicking various clinical conditions to assess biological responses to different implant conditions like geometry, surface texture, or biological cues. However, despite its robust cortical structure, certain intraosseous devices may require adaptations in design and size for successful implantation. Therefore, establishing standardized surgical methods for manipulating both soft and hard tissues in the implantation region is essential for ensuring proper implant or screw device placement, particularly in fields like implant dentistry and orthopedics. This study included eighty Sprague Dawley rats divided into two groups based on their respective diseases: Group 1 with osteoporosis and Group 2 with Type 2 Diabetes. Implantations were performed at 4 weeks and 12 weeks, with the same surgeon following a consistent surgical technique. A positive biological response was observed, indicating complete osseointegration of all implants placed. These results validate the success of the surgical protocol, which can be replicated for other studies and serve as a benchmark for the biomaterials community. Notably, osseointegration values remained stable at both 4 weeks and 12 weeks for both disease models, demonstrating a durable integration of the implant over time and emphasizing the establishment of an intimate bone connection as early as 4 weeks.

The placement of implants in a rat model is an essential experimental procedure for clinical research. This study presents a comprehensive surgical protocol for implanting titanium implants into the tibia of rat models with diabetes and osteoporosis.

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials ...