We thank Sevil Essig, Stefanie Schroth, Verena Fischer, Katja Prystaz, Yvonne H&#228;gele, and Anne Subgang for excellent technical support. We also thank the German Research Foundation (CRC1149, INST40/499-1) and the AO Trauma Foundation Germany for funding this study.

All animal experiments complied with international regulations for the care and use of laboratory animals and were approved by the regional regulatory authorities (No. 1250, Regierungspr&#228;sidium T&#252;bingen, Germany). All mice were maintained in groups of two to five animals per cage on a 14-h light, 10-h dark circadian rhythm with water and food provided ad libitum.
1. Preparation of the Surgical Material and Pre-treatment of the Mice
<ol>
	<li>Sterilize all surgical material...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+mechanisms+and+interventions.">Fracture healing: mechanisms and interventions.</a> Nat Rev Rheumatol. 11 (1), 45-54 (2015).</li><li>Augat, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+tissue+properties+in+bone+healing:+influence+of+size+and+stability+of+the+osteotomy+gap.">Local tissue properties in bone healing: influence of size and stability of the osteotomy gap.</a> J Orthop Res. 16 (4), 475-481 (1998).</li><li>Claes, L. E., Heigele, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnitudes+of+local+stress+and+strain+along+bony+surfaces+predict+the+course+and+type+of+fracture+healing.">Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing.</a> J Biomech. 32 (3), 255-266 (1999).</li><li>Claes, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+factors+on+the+fracture+healing+process.">Effects of mechanical factors on the fracture healing process.</a> Clin Orthop Relat Res. (355), 132-147 (1998).</li><li>Hu, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+to+bone+transformation+during+fracture+healing+is+coordinated+by+the+invading+vasculature+and+induction+of+the+core+pluripotency+genes.">Cartilage to bone transformation during fracture healing is coordinated by the invading vasculature and induction of the core pluripotency genes.</a> Development. 144 (2), 221-234 (2017).</li><li>Hankenson, K. D., Zimmerman, G., Marcucio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+perspectives+of+delayed+fracture+healing.">Biological perspectives of delayed fracture healing.</a> Injury. 45, 8-15 (2014).</li><li>Meyer, R. A., 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=Age+and+ovariectomy+impair+both+the+normalization+of+mechanical+properties+and+the+accretion+of+mineral+by+the+fracture+callus+in+rats.">Age and ovariectomy impair both the normalization of mechanical properties and the accretion of mineral by the fracture callus in rats.</a> J Orthop Res. 19 (3), 428-435 (2001).</li><li>Nikolaou, V. S., Efstathopoulos, N., Kontakis, G., Kanakaris, N. K., Giannoudis, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+osteoporosis+in+femoral+fracture+healing+time.">The influence of osteoporosis in femoral fracture healing time.</a> Injury. 40 (6), 663-668 (2009).</li><li>Haffner-Luntzer, M., Kovtun, A., Rapp, A. 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The author Romano Matthys is an employee of RISystem AG Davos, Switzerland that produces the implants and implant specific instruments used in this article. All other authors have no competing financial interests.

Modifications and Troubleshooting:
The main goal of this study was to describe a protocol for using of an MRI-compatible external fixator for femur osteotomy in the mouse with the ability to monitor callus tissue development longitudinally during the endochondral fracture-healing process. The design of a custom-made mounting device for insertion of the external fixator ensured a standardized position during repeated scans. Semi-automatic tissue segmentation allows the analysis...

Secondary fracture healing is the most common form of bone healing. It is a complex process mimicking specific aspects of ontogenic endochondral ossification1,2,3. The early fracture hematoma predominantly consists of immune cells, granulation and fibrous tissue. Low oxygen tension and high biomechanical strains hamper osteoblast differentiation at the fracture gap, but promote the differentiation of progenitor cells into chondrocytes4,5,6. These cells start to proliferate at the site of injury to form a cartilaginous matrix providing initial stability of the fractured bone. During callus maturation, chondrocytes become hypertrophic, undergo apoptosis, or trans-differentiate into osteoblasts. Neovascularization at the cartilage-to-bone transition zone provides elevated oxygen levels, allowing the formation of bony tissue7. After bony bridging of the fracture gap, biomechanical stability is increased and osteoclastic remodeling of the external fracture callus occurs to gain physiological bone contour and structure3. Therefore, the amounts of fibrous, cartilaginous, and bony tissue in the fracture callus provide important information about the bone healing process. Disturbed or delayed healing becomes visible by alterations of callus tissue development both in humans and mice8,9,10,11. Available in vivo techniques to longitudinally monitor callus tissue development in preclinical fracture healing studies using small animals include digital radiography and &#181;CT imaging12,13. However, both techniques are only able to discriminate between mineralized and non-mineralized tissue. In contrast, MRI provides excellent soft tissue contrast and might therefore be able to identify soft tissue and cartilage in the fracture callus.
Previous work showed promising results for post mortem MRI in mice with articular fractures14 and in vivo MRI in mice during intramembranous bone-defect healing15. However, both studies also stated limited spatial resolution and tissue contrast. We previously demonstrated the feasibility of high-resolution in vivo MRI for longitudinal assessment of soft callus formation during murine endochondral fracture healing16. Here, we report the protocol for using an MRI-compatible external fixator for femur osteotomy in mice in order to monitor callus tissue development longitudinally during the endochondral fracture healing process. The design of a custom-made mounting device for insertion of the external fixator ensured a standardized position during repeated scans.

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			<td class="xl65" height="21" style="width:254pt;height:15.75pt" width="338">Anaesthesia tube</td>
			<td class="xl65" style="width:295pt" width="393">FMI, Seeheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">ZUA-82-ANA-TUB-Mouse</td>
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			<td class="xl65" height="21" style="height:15.75pt">Anaesthetic machine&#160;</td>
			<td class="xl65">FMI, Seeheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">ZUA-82-GME-MA</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Artery forceps&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BH104R</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Autoclave</td>
			<td class="xl65">Systec, Wettenberg, Germany</td>
			<td class="xl64" style="width:149pt" width="199">DX-150</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Autoclaving packaging</td>
			<td class="xl65">Stericlin, Feuchtwangen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">2301-04/06/10/12/16</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Avizo software</td>
			<td class="xl65">FEI, Burlington, USA</td>
			<td class="xl64" style="width:149pt" width="199">-</td>
			<td class="xl67">Version 8.0.1</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">BioSpec 117/16 magnetic resonance imaging system</td>
			<td class="xl65">Bruker Biospin, Ettlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">117/16</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Bulldog clamp&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BH 021R</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Carbon steel scalpel no. 11/15</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BA211/215</td>
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			<td class="xl65" height="21" style="height:15.75pt">Ceramic mounting pin 0.45 mm&#160;</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">HS691490</td>
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			<td class="xl65" height="21" style="height:15.75pt">Clindamycin (300 mg / 2ml)</td>
			<td class="xl65">Ratiopharm, Ulm, Germany</td>
			<td class="xl64" style="width:149pt" width="199">-</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Dressing forceps 115 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BD210R</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Dressing forceps 130 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BD025R</td>
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			<td class="xl65" height="21" style="height:15.75pt">Drill bit coated 0.45 mm&#160;</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">HS820420</td>
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			<td class="xl65" height="21" style="height:15.75pt">Durogrip needle holder 125 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BM024R</td>
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			<td class="xl65" height="21" style="height:15.75pt">Foliodrape&#160;</td>
			<td class="xl65">Hartmann, Heidenheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">2513026</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Frekaderm</td>
			<td class="xl65">Fresenius, Bad Homburg, Germany</td>
			<td class="xl64" style="width:149pt" width="199">4928211</td>
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			<td class="xl65" height="21" style="height:15.75pt">Gigli saw 0.44 mm&#160;</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">RIS.590.110.25</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Hand drill</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">RIS.390.130-01</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Heating plate&#160;</td>
			<td class="xl65">FMI, Seeheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">IOW-3704</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Hygonorm gloves&#160;</td>
			<td class="xl65">Hygi, Telgte, Germany</td>
			<td class="xl64" style="width:149pt" width="199">2706</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Isoflurane</td>
			<td class="xl65">Abbot, London, UK</td>
			<td class="xl64" style="width:149pt" width="199">Forene</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Micro forceps 155 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BD343R</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Micro scissors 120 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">FD013R</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Mouse FixEx L 0.7 mm&#160;</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">RIS.611.300-10</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Needle case for drills&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BL911R</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Needle holder</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BB078R</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Octenisept</td>
			<td class="xl65">Sch&#252;lke, Norderstedt, Germany</td>
			<td class="xl64" style="width:149pt" width="199">121403</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Osirix software</td>
			<td class="xl65">Pixmeo SARL, Bernex, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">-</td>
			<td class="xl67">Version 4.0</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Oxygen, medical grade</td>
			<td class="xl65">MTI, Ulm, Germany</td>
			<td class="xl64" style="width:149pt" width="199">-</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Resolon 5/0</td>
			<td class="xl65">Resorba, N&#252;rnberg, Germany</td>
			<td class="xl64" style="width:149pt" width="199">88143</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Saline 0.9%</td>
			<td class="xl65">Braun, Melsungen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">3570350</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Scalpel handle 125 mm</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BB073R</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Scissors 150 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BC006R</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
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in vivo evaluation of fracture callus development during bone healing in mice using an mri-compatible osteosynthesis device for the mouse femur

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

First, the success of the surgical procedure can be confirmed by analysis of the MRI scans (see example in Figure 2). All four pins should be located in the middle of the femoral shaft. The size of the osteotomy gap should be between 0.3-0.5 mm. If the size of the osteotomy gap varies greatly from these values, the mouse should be excluded from further analysis.
Secondly, the evaluation of longitudi...

Watch this Scientific Journal Video about In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur at JoVE.com

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

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

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

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

The overall goal of this technique is to use magnetic resonant imaging, or MRI, for the longitudinal evaluation of murine fracture callus development during bone healing. This method can help answer key questions in the bone healing field such as how fracture calluses develop over time in different mouse models. The main advantages of this technique are that fracture callus development can be monitored in vivo in real-time and that the applied external fixator provides a highly standardized fracture healing model.Demonstrating the MRI procedure will be Anne Subgang, a technician from the Small Animal Imaging Core facility. Before beginning the procedure, confirm the appropriate level of sedation by lack of response to toe pinch and inject the mouse subcutaneously with a single dose of antibiotics in a fluid depot of two times 250 microliters of 0.9%sodium chloride. Apply ointment to the animal's eyes and place the mouse on a 37-degrees-Celsius heating pad.Remove the fur from the right hind limb and scrub the surgical area with an alcohol-based disinfectant. Place a sterile drape over the animal leaving the surgical area exposed, and cover the right hind paw with a small piece from a sterile glove. Scrub the surgical area three more times with disinfectant.Then, using an autoclave-sterilized scalpel, make an approximately one-centimeter longitudinal skin incision along the interior side of the right femur and use micro scissors and forceps to bluntly separate the biceps femoris and vastus lateralis muscles. Cut the tendon insertion at the femur trochanter to allow free access to the anterolateral part of the bone taking care that the sciatic nerve is preserved, and position the external fixator parallel to the femur. Using a 0.45-millimeter drill bit, manually drill bore holes through cortex.And starting with the most proximal and distal ends, place ceramic mounting pins into the holes. When all the pins have been placed, humidify the bone with a small volume of sterile saline and use a 0.4-millimeter Gigli wire saw to create a 0.4-millimeter osteotomy through the hole bone between the two inner pins. Flush the osteotomy gap carefully with two milliliters of sterile saline to remove any bone chips from between the two fractured cortices and use a continuous resorbable suture to adapt the muscles followed by adaptation of the skin with interrupted non-resorbable sutures.Then, clean the surgical area with disinfectant and return the mouse to its cage with monitoring until full recovery. As early as three days after the surgery, insert the external fixator carefully into a custom-made mounting device and place the anesthetized mouse onto a temperature-controlled cradle for introduction into the MRI device. Rigidly attach the external fixator to the mounting device on the four-element head coil, and use a dedicated high-field small animal MRI system operating at 11.7 tesla to apply a proton-density fat-suppressed multi-sliced turbo-spin echo sequence to the fracture.When all of the images have been acquired, open the data in the appropriate image analysis software and enter the voxel size as 0.05 by 0.05 by 0.35 millimeters cubed. To segment the different tissues in the fracture callus with semi-automatic thresholding based on their intensity, first click on the edit new label field and click Add material. Rename the material to callus, and use the lasso tool to distinguish the callus area from the surrounding tissues based on the hypointense signal from the periosteum.Click Add to material, then click Add material and rename the material to cartilage. Using the threshold tool, segment the cartilage and Select only current material from the callus. Then, click cartilage and Add to material.When all of the tissues have been segmented, click Generate surface, apply None for smoothing type and click Surface View to generate 3D reconstructions of the fractured femurs based on the tissue segmentation data. A successful surgical procedure can be confirmed by MRI scan of the fracture showing all four pins in the middle of the femoral shaft with an osteotomy gap between 0.3 and 0.5 millimeters. On day 10 postsurgery, evaluation of the longitudinal scans during the fracture healing process reveals cartilaginous tissue within the middle of the fracture callus and bony tissue at the periphery of the fracture.Both the cartilaginous and bony tissues increase until day 14. The cartilaginous tissue then decreases until day 21 at which point bony bridging can be observed. After segmentation of the different tissues in the fracture callus as just demonstrated, a whole 3D femur scan can be generated with the mature cortex appearing in gray, the ceramic pins marked in yellow, the callus soft tissue in green, the cartilage tissue in red and the callus bony tissue indicated in purple.After watching this video, you should have a good understanding of how to apply an external fixator to the mouse femur to create the femur osteotomy and to insert a mouse into an MRI device to ensure a standardized scanning position. Once mastered, the external fixator application and the osteotomy technique can be completed within 20 to 30 minutes if performed properly.

in-vivo-evaluation-fracture-callus-development-during-bone-healing

Research

JoVE Journal

Medicine

9.4K visualizações.  University Medical Center Ulm. The overall goal of this technique is to use magnetic resonant imaging, or MRI, for the longitudinal evaluation of murine fracture callus development during bone healing. This method can help answer key questions in the bone healing field such as how fracture calluses develop over time in different mouse models. The main advantages of this technique are that fracture callus development can be monitored in vivo in real-time and that the applied external fixator provides a highly standardized f...