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. Use an autoclaving temperature of 120-135 &#176;C for 20-30 min of sterilization time.</li>
	<li>Purchase C57BL/6 mice or mice from another strain which are between 19-35 g of body weight. Follow the appropriate animal care and experimental protocols in accordance with national guidelines that is approved by the investigator&#39;s Institutional Animal Care and Use Committee. Allow a minimum of 7 days acclimatization period before starting the procedure.</li>
	<li>Provide analgesia to all mice via the drinking water one day before surgery until the third postoperative day.</li>
</ol>
2. Surgical Procedure and Application of the External Fixator
<ol>
	<li>Place the mouse into a tube preloaded with 5-7% isoflurane and 60 mL/min oxygen. After loss of postural reflexes, remove the mouse from the anesthesia induction tube and maintain the anesthesia via an inhalation mask providing 1-3% isoflurane and 60 mL/min oxygen.
	<ol>
		<li>Monitor the breathing pattern and hind paw reflex during anesthesia. Ensure that the breathing rate is around 100 cycles/min and the hind paw reflex is absent. 
		&#8203;NOTE: The amount of gas needed is dependent on age, sex, body weight, and strain of the mouse.</li>
	</ol>
	</li>
	<li>Prior to surgery, inject the mouse with a single dose of antibiotics subcutaneously (clindamycin, 45 mg/kg). Furthermore, for maintenance of the physiological fluid balance, inject the mouse with a subcutaneous fluid depot of 500 &#181;L saline (0.9% NaCl).</li>
	<li>To prevent corneal drying, apply eye ointment to the mouse eyes. Place the mouse on a heating plate at 37 &#176;C during the anesthesia and surgical procedure to maintain physiological body temperature.</li>
	<li>Remove the fur from the right hind limb and scrub the surgical area with an alcohol-based disinfectant. Cover the right hind paw with a small part of a sterile glove to avoid unsterile areas. Disinfect the right hind limb three times. Place a sterile drape over the whole mouse except for the surgical area.</li>
	<li>Incise the skin approximately 1 cm longitudinally along the anterior side of the right femur with a scalpel. Separate bluntly the m. biceps femoris and the m. vastus lateralis with micro scissor and forceps. Cut the tendon origin&#160;side at the femur trochanter with a micro scissor to allow free access to the anterolateral part of the bone. Make sure that the sciatic nerve is preserved.</li>
	<li>Position the external fixator (axial stiffness of 3 N/mm, Figure 1A) parallel to the femur. Manually drill the boreholes through cortex with a 0.45-mm drill bit and place the ceramic mounting pins into the boreholes. Start with the most proximal pin, followed by the most distal pin, and the two pins in between.
	<ol>
		<li>Make sure that there is no tension, compression, or shear stress on the fixator during the mounting procedure, otherwise the achieved osteotomy gap will not be sufficient due to relaxation of the fixator.</li>
	</ol>
	</li>
	<li>Humidify the bone with a small amount of sterile NaCl to avoid dehydration during the sawing procedure.</li>
	<li>Create a 0.4-mm osteotomy through the whole bone between the two inner pins by using a 0.4 mm gigli wire saw. 
	NOTE: Optionally, an oscillating micro saw can be used to create the osteotomy. Make sure to avoid any metal chips from the saw at the osteotomy area.</li>
	<li>Flush the osteotomy gap carefully with 2 mL of sterile NaCl to remove bone chips between the two fractured cortices.</li>
	<li>Adapt the muscles by using a continuous suture with a resorbable suture (see Table of Materials). Then adapt the skin by using interrupted non-resorbable sutures (see Table of Materials). To avoid wound biting, do not place the suture at the cranial part of the wound. 
	NOTE: Do not use skin glue or clips since mice usually remove it from the wound causing further damage to the skin.</li>
	<li>Clean the surgical area with a disinfectant and place the mouse into its cage.&#160;Monitor the mouse and supply sufficient heat (e.g. by infrared light) until it is fully awake.&#160;Monitor water, food intake, and body weight after the surgery to make sure the animal is not in pain and distress. Provide analgesia to all mice via the drinking water until the third postoperative day. 
	NOTE: Mice may be housed in groups of up to four animals.</li>
	<li>Monitor the mouse&#39;s activity on days 1 to 5 after surgery. During that time course, the mouse should bear weight on the operated limb. Otherwise, the mouse must be excluded from further analysis.</li>
</ol>
3. MRI Procedure and Image Analysis
<ol>
	<li>Prior to the MRI scanning procedure, anaesthetize the mouse according to the protocol in steps 2.1 and 2.3, and keep the respiratory rate around 100 cycles/min. Insert the external fixator at the right hind limb of the mouse carefully into a custom-made mounting device (Figure 1B, C).
	<ol>
		<li>Make sure to avoid bending or compression of the fixator during this step since this may interfere with fracture healing. 
		Note: The MRI scans can be conducted as early as 3 days after surgery, depending on the animal care and experimental protocol.</li>
	</ol>
	</li>
	<li>Place the mouse on a temperature controlled cradle for introduction into the MRI device. Attach the mounting device rigidly to the four-element head coil.</li>
	<li>Acquire MRI data using a dedicated high-field small-animal MRI system operating at 11.7 T. 
	&#8203;NOTE: The MRI data acquisition geometry is aligned with the femur bone, orthogonally to the screws.
	<ol>
		<li>Acquire data by applying a proton-density fat-suppressed multi-slice TSE sequence (PD-TSE) using acquisition parameters: echo/repetition time TE = 5.8 ms/TR = 2,500 ms, resolution &#916;r = 52 &#215; 52 &#215; 350 &#181;m&#179;, field-of-view (FOV) = 20 &#215; 20 mm&#178;, and bandwidth &#916;&#969;= 150 KHz.</li>
		<li>NOTE: The total acquisition time for 22 slices is 36 min.</li>
	</ol>
	</li>
	<li>Open the acquired data with image analysis software. Enter the voxel size as 0.05 x 0.05 x 0.35 mm3. Segment the different tissues in the fracture callus (bone, cartilage, fibrous tissue/bone marrow) based on their intensity with semi-automatic thresholding as follows.
	<ol>
		<li>Click the &#34;Edit New Label Field&#34;, click &#34;Add Material&#34;, and rename the material to &#34;callus&#34;. Distinguish the callus area from the surrounding tissues based on the hypo-intense signal from the periosteum using the &#34;Lasso&#34; tool.</li>
		<li>Click &#34;add to material&#34;. Click &#34;Add Material&#34; and rename the material to &#34;cartilage&#34;. Segment the cartilage by using the &#34;threshold&#34; tool and &#34;Select only current material&#34; from &#34;callus&#34;. Click &#34;cartilage&#34; and &#34;add to material&#34;. Repeat these steps with &#34;bone&#34; and &#34;bone marrow/fibrous tissue&#34;.</li>
	</ol>
	</li>
	<li>Generate 3D reconstructions of the fractured femurs based on the tissue segmentation data using image analysis software. Click &#34;Generate Surface&#34;, apply &#34;None&#34; for &#34;Smoothing Type&#34; and click &#34;Surface View&#34;. 
	NOTE: Very small, hyper-intense areas surrounding the ends of the fractured cortices are likely to be artifacts due to the transition from bony to soft tissue. These areas should be excluded from further analysis. Hyper-intense areas in the middle of the fracture callus during the endochondral phase of fracture healing represent cartilaginous tissue. Hypo-intense areas at the fracture callus distal from the osteotomy gap at the endochondral ossification phase and areas with the same intensity throughout the whole fracture callus at later healing stages represent newly formed bony callus tissue. Although these areas have a hypo-intense signal, the signal intensity from mature bone (cortex) is even lower. After thresholding the signal intensity for bony tissue and cartilaginous tissue in the fracture callus, mark the remaining tissue as bone marrow and fibrous tissue. Values for tissue segmentation are: bony tissue (including mature cortex, trabecular bone, and bony callus tissue) is segmented within the range of 1-3.3 (normalized signal intensity to mature cortex), bone marrow/fibrous tissue within the range of 3.4-5.4, and cartilaginous callus tissue within the range of 5.5-6.2.</li>
	<li>If needed, repeat the MRI scan longitudinally during the fracture healing process. To monitor cartilaginous callus development, scan the mice on days 10, 14, and 21 after surgery. 
	NOTE: The time points may depend on the animal care and experimental protocol.</li>
</ol>

<ol>
	<li>Claes, L., Recknagel, S., Ignatius, A. <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+under+healthy+and+inflammatory+conditions.">Fracture healing under healthy and inflammatory conditions.</a> Nat Rev Rheumatol. 8 (3), 133-143 (2012).</li><li>Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+and+molecular+biology+of+fracture+healing.">The cell and molecular biology of fracture healing.</a> Clin Orthop Relat Res. (355), S7-S21 (1998).</li><li>Einhorn, T. A., Gerstenfeld, L. 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. E., Ignatius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+in+Bone+Fracture+Healing+Research.">Mouse Models in Bone Fracture Healing Research.</a> Current Molecular Biology Reports. 2 (2), 101-111 (2016).</li><li>Garcia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+animal+models+of+delayed+bone+healing+and+non-union+formation:+a+comprehensive+review.">Rodent animal models of delayed bone healing and non-union formation: a comprehensive review.</a> Eur Cell Mater. 26, 1-14 (2013).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+bone+healing+models:+standards,+tips,+and+pitfalls+results+of+a+consensus+meeting.">Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting.</a> Bone. 49 (4), 591-599 (2011).</li><li>Zachos, T. A., Bertone, A. L., Wassenaar, P. A., Weisbrode, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+the+study+of+articular+fracture+healing.">Rodent models for the study of articular fracture healing.</a> J Invest Surg. 20 (2), 87-95 (2007).</li><li>Taha, M. 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=Assessment+of+the+efficacy+of+MRI+for+detection+of+changes+in+bone+morphology+in+a+mouse+model+of+bone+injury.">Assessment of the efficacy of MRI for detection of changes in bone morphology in a mouse model of bone injury.</a> J Magn Reson Imaging. 38 (1), 231-237 (2013).</li><li>Haffner-Luntzer, M., 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=Evaluation+of+high-resolution+In+Vivo+MRI+for+longitudinal+analysis+of+endochondral+fracture+healing+in+mice.">Evaluation of high-resolution In Vivo MRI for longitudinal analysis of endochondral fracture healing in mice.</a> PLoS One. 12 (3), 0174283 (2017).</li><li>Beckmann, N., Falk, R., Zurbrugg, S., Dawson, J., Engelhardt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+infiltration+into+the+rat+knee+detected+by+MRI+in+a+model+of+antigen-induced+arthritis.">Macrophage infiltration into the rat knee detected by MRI in a model of antigen-induced arthritis.</a> Magn Reson Med. 49 (6), 1047-1055 (2003).</li><li>Al Faraj, ., Shaik A, S. u. l. t. a. n. a., Pureza, A., A, M., Alnafea, M., Halwani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+macrophage+recruitment+and+polarization+in+LPS-induced+animal+model+for+COPD:+noninvasive+tracking+using+MRI.">Preferential macrophage recruitment and polarization in LPS-induced animal model for COPD: noninvasive tracking using MRI.</a> PLoS One. 9 (3), 90829 (2014).</li><li>Rolle, A. M., 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=ImmunoPET/MR+imaging+allows+specific+detection+of+Aspergillus+fumigatus+lung+infection+in+vivo.">ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo.</a> Proc Natl Acad Sci U S A. 113 (8), 1026-1033 (2016).</li><li>Niemeyer, M., 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=Non-invasive+tracking+of+human+haemopoietic+CD34(+)+stem+cells+in+vivo+in+immunodeficient+mice+by+using+magnetic+resonance+imaging.">Non-invasive tracking of human haemopoietic CD34(+) stem cells in vivo in immunodeficient mice by using magnetic resonance imaging.</a> Eur Radiol. 20 (9), 2184-2193 (2010).</li></ol>

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.

<table><tbody><tr><td>Anaesthesia tube</td><td>FMI, Seeheim, Germany</td><td>ZUA-82-ANA-TUB-Mouse</td><td></td></tr><tr><td>Anaesthetic machine&amp;nbsp;</td><td>FMI, Seeheim, Germany</td><td>ZUA-82-GME-MA</td><td></td></tr><tr><td>Artery forceps&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BH104R</td><td></td></tr><tr><td>Autoclave</td><td>Systec, Wettenberg, Germany</td><td>DX-150</td><td></td></tr><tr><td>Autoclaving packaging</td><td>Stericlin, Feuchtwangen, Germany</td><td>2301-04/06/10/12/16</td><td></td></tr><tr><td>Avizo software</td><td>FEI, Burlington, USA</td><td>-</td><td>Version 8.0.1</td></tr><tr><td>BioSpec 117/16 magnetic resonance imaging system</td><td>Bruker Biospin, Ettlingen, Germany</td><td>117/16</td><td></td></tr><tr><td>Bulldog clamp&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BH 021R</td><td></td></tr><tr><td>Carbon steel scalpel no. 11/15</td><td>Aesculap, Tuttlingen, Germany</td><td>BA211/215</td><td></td></tr><tr><td>Ceramic mounting pin 0.45 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>HS691490</td><td></td></tr><tr><td>Clindamycin (300 mg / 2ml)</td><td>Ratiopharm, Ulm, Germany</td><td>-</td><td></td></tr><tr><td>Dressing forceps 115 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD210R</td><td></td></tr><tr><td>Dressing forceps 130 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD025R</td><td></td></tr><tr><td>Drill bit coated 0.45 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>HS820420</td><td></td></tr><tr><td>Durogrip needle holder 125 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BM024R</td><td></td></tr><tr><td>Foliodrape&amp;nbsp;</td><td>Hartmann, Heidenheim, Germany</td><td>2513026</td><td></td></tr><tr><td>Frekaderm</td><td>Fresenius, Bad Homburg, Germany</td><td>4928211</td><td></td></tr><tr><td>Gigli saw 0.44 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>RIS.590.110.25</td><td></td></tr><tr><td>Hand drill</td><td>RISystem, Davos, Switzerland</td><td>RIS.390.130-01</td><td></td></tr><tr><td>Heating plate&amp;nbsp;</td><td>FMI, Seeheim, Germany</td><td>IOW-3704</td><td></td></tr><tr><td>Hygonorm gloves&amp;nbsp;</td><td>Hygi, Telgte, Germany</td><td>2706</td><td></td></tr><tr><td>Isoflurane</td><td>Abbot, London, UK</td><td>Forene</td><td></td></tr><tr><td>Micro forceps 155 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD343R</td><td></td></tr><tr><td>Micro scissors 120 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>FD013R</td><td></td></tr><tr><td>Mouse FixEx L 0.7 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>RIS.611.300-10</td><td></td></tr><tr><td>Needle case for drills&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BL911R</td><td></td></tr><tr><td>Needle holder</td><td>Aesculap, Tuttlingen, Germany</td><td>BB078R</td><td></td></tr><tr><td>Octenisept</td><td>Sch&amp;uuml;lke, Norderstedt, Germany</td><td>121403</td><td></td></tr><tr><td>Osirix software</td><td>Pixmeo SARL, Bernex, Switzerland</td><td>-</td><td>Version 4.0</td></tr><tr><td>Oxygen, medical grade</td><td>MTI, Ulm, Germany</td><td>-</td><td></td></tr><tr><td>Resolon 5/0</td><td>Resorba, N&amp;uuml;rnberg, Germany</td><td>88143</td><td></td></tr><tr><td>Saline 0.9%</td><td>Braun, Melsungen, Germany</td><td>3570350</td><td></td></tr><tr><td>Scalpel handle 125 mm</td><td>Aesculap, Tuttlingen, Germany</td><td>BB073R</td><td></td></tr><tr><td>Scissors 150 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BC006R</td><td></td></tr><tr><td>Sealer for autoclave packaging&amp;nbsp;</td><td>Hawo GmbH, Obrigheim, Germany</td><td>HM500</td><td></td></tr><tr><td>Sterican 27 G&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>4657705</td><td></td></tr><tr><td>Sterile surgical blades no. 11/15&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BB511/515</td><td></td></tr><tr><td>Surgical gloves&amp;nbsp;</td><td>Hartmann, Heidenheim, Germany</td><td>Peha-micron 9425712</td><td></td></tr><tr><td>Surgical light&amp;nbsp;</td><td>Maquet SA, Ardon, France</td><td>Blue line 80</td><td></td></tr><tr><td>Syringes 5 ml&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>Injekt 4606051V</td><td></td></tr><tr><td>Tissue forceps 80 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>OC091R</td><td></td></tr><tr><td>Tramadol 25 mg/l</td><td>Gr&amp;uuml;nenthal, Aachen, Germany</td><td>100mg/ml</td><td></td></tr><tr><td>Vasofix Safety&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>4268113S-01</td><td></td></tr><tr><td>Vicryl 5-0&amp;nbsp;</td><td>Ethicon, Norderstedt, Germany</td><td>V30371</td><td></td></tr><tr><td>Visdisic eye ointment&amp;nbsp;</td><td>Bausch &amp;amp; Lomb, Berlin, Germany</td><td>3099559</td><td></td></tr></tbody></table>

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 ...

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

Research

JoVE Journal

Medicine

9.5K Views.  University Medical Center Ulm. De evaluatie van de ontwikkeling van het weefsel in de breuk eelt tijdens het endochondral bot genezing is essentieel voor het controleren van het genezingsproces. Hier, rapporteren we het gebruik van een magnetische resonantie imaging (MRI)-compatibele externe fixator voor de muis dijbeen toe MRI scans tijdens bot regeneratie in muizen.

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

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Developmental Biology

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

The 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 ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

Biology

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

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

Samenvatting

Meer video's verkennen