The authors wish to thank Rena Bizios for her valuable comments on the manuscript.

Ethics Statement: The mice used in the present study were treated in accordance with the guidelines published by the European Committee for &#34;Care and Use of Laboratory Animals&#34; (Directive 2010/63/EU and the European Convention ETS 123). The experimental protocol was approved by the Ethics Committee of the Faculty of Medicine Lariboisi&#232;re Saint-Louis (CEEA LV/2010-01-04).
1. Animals
<ol>
	<li>Use athymic mice (10 weeks old). Use a minimal number of 6 mice with defect left empty as negative control group.</li>
</ol>
2. Scaffolds Preparation
<ol>
	<li>Syngenic Graft Preparation
	<ol>
		<li>Use bone isograft to fill the defect to provide control group with a minimum number of 6 animals.</li>
		<li>Obtain bone isografts by harvesting excised femoral bone from mouse belonging to either &#34;defects left empty or &#34;defects filled with coral scaffold&#34; groups (this avoids the use of extra animal to collect bone isograft)11.</li>
		<li>Flush the resected bone with Phosphate Buffered Saline (PBS) and keep it sterile using moist gauze compress.</li>
	</ol>
	</li>
	<li>Coral Scaffold Preparation
	<ol>
		<li>Use scaffold that are made of natural coral: Acropora sp. coral exoskeleton cubes, 3 x 3 x 3 mm as potential bone substitute with a minimum of 6 animals.</li>
		<li>Carve by hand each coral cube to the shape of cylinder (3.5 height; 2 mm diameter).</li>
		<li>Sterilize each scaffold by autoclaving (121 &#176;C for 20 min), wash it with sterile PBS, and immersed it in complete culture medium (&#945;-MEM) for 24 hr before implantation in mice.</li>
	</ol>
	</li>
</ol>
3. Anesthetic Procedures and Analgesia
<ol>
	<li>Provide preventive analgesia, 15 min prior to anesthesia, by subcutaneous injection of buprenorphine (0.1 mg/kg animal body weight).</li>
	<li>Apply ointment in the animal eyes to prevent dryness every 30 min while the animals are under anesthesia.</li>
	<li>Place the mice on a warming pad to prevent hypothermia.</li>
	<li>Anesthesia and Analgesia during the Surgical Procedure
	<ol>
		<li>Inject intraperitoneally a solution containing xylazine (8 mg/kg) and ketamine (100 mg/kg).</li>
		<li>Deliver oxygen via flow-by (50 ml/min).</li>
		<li>Confirm adequate depth of anesthesia by the presence of good muscle relaxation and lack of animal response to a noxious stimulus (e.g., firm toe pinch).</li>
		<li>Inject subcutaneously a single dose of enrofloxacin (0.05 mg/kg) as microbial prophylaxis.</li>
	</ol>
	</li>
	<li>Post-operative Analgesia
	<ol>
		<li>Provide postoperative analgesia by subcutaneous injection of buprenorphine (0.1 mg/kg) every 12 hr for 3 consecutive days.</li>
	</ol>
	</li>
	<li>Anesthesia during Diagnostic Imaging Procedures
	<ol>
		<li>Place the mice in an anesthetizing-box, and then induce and maintain anesthesia using approximately 4% and 2% isoflurane in oxygen, respectively.</li>
		<li>Confirm adequate depth of anesthesia by good animal muscle relaxation and the lack of movement.</li>
	</ol>
	</li>
	<li>Recovery Conditions
	<ol>
		<li>Keep the mice on warming pad until full recovery</li>
		<li>Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency after surgery.</li>
		<li>Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	</ol>
	</li>
	<li>Post-operative Conditions
	<ol>
		<li>Host the mice separately during the first 3 days Host the mice by 4 in cages after day 3.</li>
		<li>Provide water and adapted food ad libitum. Allow the mice to weight-bear, without any activity restriction throughout the post-operative period.</li>
	</ol>
	</li>
</ol>
4. Surgical procedure: Femoral Segmental Defect Model11,12
<ol>
	<li>After anesthesia, place each mouse in ventral recumbency with the left hind limb in extension.</li>
	<li>Scrub the limb for aseptic surgery using 10% povidone iodine for 5 min and then place a sterile drape under the limb to create a sterile surface (a sterile transparent drape is used in order to be able to monitor respiratory movement during the procedure). Care is taken to maintain sterility of the surgical field during the procedure.</li>
	<li>Make a 15 - 17-mm longitudinal skin incision over the anterolateral aspect of the femur, extending from the hip joint to the stifle joint.</li>
	<li>Incise the fascia lata, split the vastus lateralis muscle and the biceps femoris muscle to expose the full length of the femoral diaphysis. Caution should be taken to preserve the sciatic nerve caudally and the articular capsule distally (Figure 1).</li>
	<li>To enhance femoral diaphysis exposure, transect the gluteal muscle and biceps femoris from the 3rd trochanter.</li>
	<li>Perform a circular dissection of the femur at the middle of the diaphysis.</li>
	<li>Apply a 6-hole titanium micro-locking plate (10 mm long; 1.5 mm wide, weight: 30 mg) on the anterior femoral side. 
	NOTE: The holes of the plate, which are conically recessed with a cylindrical portion, accommodate titanium self-tapping locking screws (2 mm long, 0.47 mm outer diameter, weight: 5 mg, with undersurface of the head screw threaded to enable locking within the plate hole) that are connected to a stem, which twists off when locked.</li>
	<li>Drill the most proximal hole of the plate using a 0.3 mm drill bit and either dedicated engine power or non-dedicated engine power operated at 2,500 rpm at approximately 500 mW12).</li>
	<li>Insert the first screw using a dedicated screwdriver and then lock it (Figure 2). 
	NOTE: Since alignment of the plate is determined by application of this first screw, it is important to position the plate parallel to the femur when inserting the screw.</li>
	<li>Drill the most distal hole of the plate in a similar fashion, insert and lock the screw (Figure 3).</li>
	<li>Insert, but do no lock, the two other outer screws.</li>
	<li>Place the wire of the 0.22 mm Gigli saw closely around the bone in a medio-lateral orientation and then insert it in the slots of the jig (Figure 4).</li>
	<li>Insert the dedicated jig on the stem of the two last screws and apply it above the plate (Figure 5).</li>
	<li>Perform a 3.5-mm long mid-diaphyseal femoral ostectomy using the Gigli saw under irrigation (using sterile isotonic saline) to prevent thermal necrosis. Have the surgeon&#39;s assistant take the jig. Have the surgeon apply a constant steady tension. Be careful not to tangle the saw wire and to use the middle two-thirds of the wire. Avoid excess movement in order to obtain a straight bone cut (Figure 6).</li>
	<li>After the ostectomy, remove the Gigli saw. To avoid damage of the soft tissue, cut the saw wire close to the bone on one side.</li>
	<li>Remove the jig and lock the two last screws (Figure 7).</li>
	<li>Either leave the segmental defect empty or surgically fill it by putting materials to be tested inside the defect.</li>
	<li>Copiously rinse the surgical field with sterile isotonic saline.</li>
	<li>Place the vastus lateralis muscle loosely over the plate. Close the fascia and subcutaneous planes using a simple continuous suture pattern and 5.0 glycomer 631 suture; close the skin with a simple interrupted suture pattern using 4.0 glycomer 631 suture. Alternatively, it is also possible to close the skin using skin glue.</li>
</ol>
5. In Vivo Assessments of Bone Regeneration
<ol>
	<li>With the mice under anesthesia, perform radiographic assessments in a longitudinal manner using both conventional X-rays (26 kV, 10 sec; 2X magnification; 20 lines/mm spatial resolution) and high-resolution micro-computed tomography (&#956;CT).</li>
	<li>For &#956;CT analysis, acquire images at a resolution of 36 &#956;m (50 kV and 478 mA, at 40 msec exposure time, using a 0.5 mm aluminum filter, rotation step of 0.7&#186;, and tomographic rotation of 180&#186;). Analyze the images using the resident software.</li>
</ol>
6. Ex Vivo Assessments of Bone Regeneration
<ol>
	<li>Ten weeks after surgery, induce anesthesia using isoflurane in oxygen, and then sacrifice the mice by intraperitoneal injection of an overdose of barbiturate (1 ml of pentobarbital).</li>
	<li>Excise the femoral bones, remove overlaying muscle tissue, and fix the bone specimens in 4% paraformaldehyde (pH 7.4) for four days.</li>
	<li>Remove the plate and screws from each excised bone specimen after paraformaldehyde fixation.</li>
	<li>Ex Vivo &#956;CT Analysis
	<ol>
		<li>Place each excised and fixed bone in polyethylene tubes filled with 75% alcohol and analyze it using ex vivo &#956;CT.</li>
		<li>Acquire images at 80 kV and 100 &#956;A (exposure time of 1,000 msec, aluminum 0.5 filter, and 4 &#956;m camera pixel size (2,400 x 4,000 with a voxel size of 7 &#956;m), average four frames for each rotation increment of 0.9&#186;.</li>
		<li>Reconstruct 3-dimensional images (average voxel size of 13 &#956;m) using a Hamming-filtered back-projection with the resident software.</li>
		<li>For quantitative analysis of bone formation, use resident software to obtain the volume of mineralized tissue (lower grey threshold of 45 grayscale indices and upper grey threshold of 240 grayscale indices) in a determined and consistent region of interest corresponding to the defect.</li>
		<li>Perform analyses in the same manner for each mouse with the same region of interest.</li>
		<li>Use the one-way analysis test (confidence interval - at 95% and the significant level at p &#60; 0.05) to compare the bone union rate and the volume of mineralized tissue in the region of interest between groups.</li>
	</ol>
	</li>
	<li>Histological Analysis
	<ol>
		<li>Embed each excised and fixed femoral bone in methyl methacrylate resin and process it for undecalcified histology.</li>
		<li>Cut each bone specimen lengthwise into thick section (200 &#956;m) using a circular water-cooled diamond saw.</li>
		<li>Grind each bone specimen section down to a thickness of 100 &#956;m, polish it, and stain it using Stevenel blue and van Gieson picrofuchsin stains. 
		NOTE: After staining, cells appear in blue, bone in pink, and coral in brown under light microscopy.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Ectopic implantation of orthopaedic-related materials and device in mice is commonly performed to assess the bone forming capacities of various scaffolds13,14. Important differences however exist between ectopic and orthotopic models, including native osteogenic signaling factors and paracrine interactions with host bone-forming cells.
The present study establishes a reproducible murine large segmental, critical-size femoral defect (3.5 mm, approximately 20-25% of the femur length). Considering the size of such defect and the stability provided by the resultant plate osteosynthesis, this model mimics the clinically-encountered atrophic bone non-union.
The post-operative time period chosen in the present study, is in line with previously described non-union models mice, showing a lack of adequate healing after 8 to 12 weeks4,9,15,16.
Most importantly, reproducible and stable osteosynthesis, as well as stability of the implanted bone substitutes were obtained without significant morbidity and mortality1,2 with the use of both locking plate and a jig to perform the ostectomy. This outcome contrasts also the results reported when either an external fixator or an intramedullary nail were used 4,5,17-24. For the external fixators potential disadvantages include: variability in stiffness, infections of the pins tracts, loosening of the pins, potentials injuries due to the pins and the weight of the materials (4 to 20% of the mouse body weight). For the intramedullary nail potential disadvantages include: filling of the medullary cavity with the nail and iatrogenic damage of the articular surfaces.
Other murine segmental, critical-size femoral defects stabilized by plate osteosynthesis have been described with bone defect created by a burr and ranging from 1.5 to 2-mm length16,25. In the present model, the use of a jig and a saw wire allowed a precise 3.5 mm-long ostectomy without significant muscles trauma.
However, to succeed in performing the procedure one should take on consideration several key points: Do not use small mice (Nude mice with either a weight under 25 g or age under 8 weeks) otherwise the plate should be too long. When approaching the femoral bone, take care to preserve both the sciatic nerve caudally and the articular capsule distally. Apply the plate on the anterior side of the femoral bone and since alignment of the plate is determined by application of this first screw, take care to position the plate parallel to the femur when inserting this first screw.
Before making the ostectomy, take care to perform a circular dissection of the femur at the middle of the diaphysis to avoid muscular trauma. When performing the ostectomy, the surgeon&#39;s assistant must hold firmly the guide and the surgeon must be careful (i) not to tangle the saw wire, (ii) to use the middle two-thirds of the wire while applying a constant steady tension, and (iii) to avoid excess movement to obtain a straight bone cut.
Bone healing is possible in the present model provided a bone graft is used. Moreover, this model allows further studies of the mechanisms involved in bone replacement strategies when either human-origin grafts or cells are used in a well-standardized, large, segmental, bone defect.
In addition, in line to current trends requiring refinement and reduction of use of animals in orthopedics-related research, this model can be used in conjunction with in vivo imaging techniques such as bioluminescence. Such non-invasive techniques allow monitoring both implanted cell survival and tissue healing without requiring animal sacrifice 26.
Major limitations of the present model are both the load-bearing conditions and the volume of the bone defect created because they do not fully mimic those encountered clinically in humans. Other limitations of the model are (i) the radio-opacity of the plate which may require removal of the plate before ex vivo &#956;CT analysis and may complicate interpretation of the longitudinal radiographic examination results and, (ii) the inability to modulate plate stiffness which may be a key mechanical parameter in bone formation27-30.
One must keep in mind also, when using either bone isograft or other scaffolds containing a mineral component (specifically calcium carbonate), that some bias are introduced in the segmentation process of the micro-CT analysis, because newly-formed bone density partly overlapped with either the isograft density or scaffold density. For this reason the bone volume obtain by the micro-CT analysis mostly reflect the volume of mineralized tissue (newly-formed bone plus bone substitute)11,26,31.

Massive diaphyseal bone defects are a great challenge to the orthopaedic surgeon. Bone replacement with autologous bone graft, currently considered as the gold-standard treatment, is in limited supply and is associated with harvesting-related morbidity. For these reasons, tissue-engineered bone constructs combining bone marrow mesenchymal stem cells with osteoconductive scaffolds have been explored as an alternative for autografts in orthopaedic surgery.
To date, most of the studies have been performed in clinically-relevant animal models such as dogs, pigs and sheep1-3, but preliminary evaluation of these constructs in orthotopic, segmental, critical-size bone defects in small-animal models (like mice) could have several advantages: (i) low expenses, (ii) large numbers of animals can be operated; (iii) in contrast to large animal models, homogeneity of the mouse strains limits individual variations in scaffold resorption and bone formation and; (iv) most importantly, availability of specific antibodies and gene-targeted animals enable the evaluation of the biological process involved in bone healing. Last but not least, use of immunodeficient strains of mice also enables studies using either grafts or cells of human origin without adverse immune responses in mice.
Despite the aforementioned advantages, massive diaphyseal bone defect models in mice are sparse. Most of such models use bone fixation with an intramedullary pin which fills the bone marrow cavity (thus limiting the volume of material to be tested) and also impedes reproducibility by not providing rotational and axial stability2,4-7.
The objectives of the current study are (i) mimicking a clinical bone non-union situation, to describe a reproducible, critical-size, segmental, femoral defect model in mice, which is stabilized by accurate and reproducible locking-plate osteosynthesis that provides a highly stable biomechanical environment8-10; (ii) to illustrate the present model with two potential bone substitutes and to describe bone formation analyses that could be used.

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			<td height="21" style="height:21px;width:335px;">&#945;-MEM , Minimum Essential Medium Eagle</td>
			<td style="width:191px;">Sigma-Aldrich, France</td>
			<td style="width:119px;">M4526</td>
			<td style="width:167px;">500 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acropora sp. coral exoskeleton cubes, Biocoral&#174;</td>
			<td>Biocoral&#174;, Inoteb, France</td>
			<td style="width:119px;"></td>
			<td>3x3x3 mm cubes, autoclaving (121&#176;C for 20 min) sterilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine, Buprecare&#174;</td>
			<td>Axience, Pantin, France</td>
			<td style="width:119px;"></td>
			<td>0.3 mg/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Xylazine, Rompun&#174; 2%</td>
			<td colspan="2">Bayer HealthCare, Puteaux, France</td>
			<td>20 mg/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ketamine, Ketamine 500&#174;</td>
			<td>Virbac, Carros, France</td>
			<td style="width:119px;"></td>
			<td>50 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane, For&#232;ne&#174;</td>
			<td>Abbott, Arcueil, France</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Enrofloxacine, Baytril&#174; 5%</td>
			<td colspan="2">Bayer HealthCare, Puteaux, France</td>
			<td>50 mg/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital, Dolethal&#174;&#160;</td>
			<td>V&#233;toquinol, Lure, France</td>
			<td style="width:119px;"></td>
			<td>182,2 mg/ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anesthetizing box</td>
			<td>Ugo Basile, Gemonio, Italy</td>
			<td style="width:119px;">7900/10</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Plastic transparent sterile drape, BusterOpCover 30*45cm</td>
			<td>Buster, Coveto, Montagu, France</td>
			<td style="width:119px;">613867</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">10% povidone iodine, V&#233;t&#233;dine&#174; Solution</td>
			<td style="width:191px;">V&#233;toquinol, Lure, France</td>
			<td style="width:119px;"></td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Titanium micro- locking plate, MouseFix Plate XL</td>
			<td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td>
			<td style="width:119px;">RIS.401.120</td>
			<td>6 holes, 10 mm long and 1.5 mm wide, autoclaving (121&#176;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td>
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			<td height="21" style="height:21px;">0.3 mm drill bit, Drill Bit 0.30 mm</td>
			<td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td>
			<td style="width:119px;">RIS.592.200</td>
			<td>autoclaving (121&#176;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td>
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			<td height="21" style="height:21px;">Engine power</td>
			<td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td>
			<td style="width:119px;">AccuPen</td>
			<td>Cold sterilzation (ethylene oxide)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Screw driver, Handrill</td>
			<td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td>
			<td style="width:119px;">RIS.390.130</td>
			<td>autoclaving (121&#176;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Self-tapping locking screws, MouseFix Screw 2 mm</td>
			<td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td>
			<td style="width:119px;">RIS.401.100</td>
			<td>2 mm long, 0.47 mm outer diameter and 0.34 mm core diameter, autoclaving (121&#176;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Jig,MouseFix XL Drill and Saw Guide</td>
			<td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td>
			<td style="width:119px;">RIS.301.103</td>
			<td>3.5 mm between the slots, autoclaving (121&#176;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22-mm Gigli saws (0.22 mm Saws)</td>
			<td colspan="2">RISystem AG, Davos, Switzerland</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">5.0 glycomer 631, Biosyn</td>
			<td style="width:191px;">Covidien, V&#233;toquinol, Lure, France</td>
			<td style="width:119px;"></td>
			<td>Tapper-cut needle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">4.0 glycomer 631, Biosyn</td>
			<td style="width:191px;">Covidien, V&#233;toquinol, Lure, France</td>
			<td style="width:119px;"></td>
			<td>Tapper-cut needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Xray, MX20</td>
			<td colspan="3">Faxitron X-ray Corp, Edimex, Le Plessis Grammorie</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:335px;">in vivo high-resolution microcomputed tomography, Skyscan 1176</td>
			<td colspan="2">Skyscan, Aartselaar, Belgium</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Ex vivo high-resolution microcomputed tomography, Skyscan 1172</td>
			<td colspan="2">Skyscan, Aartselaar, Belgium</td>
			<td></td>
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			<td height="21" style="height:21px;width:335px;">Resident software: Nrecon(v1.6.9)/Ctan(v.1.14.4)</td>
			<td colspan="2">Skyscan, Aartselaar, Belgium</td>
			<td></td>
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establishment of a segmental femoral critical-size defect model in mice stabilized by plate osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

The aforementioned surgical procedures lasted from 45 to 60 min. Ostectomy and osteosynthesis were easy to perform with the help of a surgeon&#39;s assistant but without using any magnifying system. No intraoperative complications occurred. In a preliminary study on 18 mice11, postoperative radiographs provided evidence that the bone defect length (3.43&#160;&#177; 0.12 mm) and the plate positioning (distance between the stifle joint cavity and the distal part of plate = 2.65&#160;&#177; 0.56 mm) were reproducible.
The anesthesia-related mortality rate was about 5%.
Functional recovery of the operated limb was excellent in all animals and full weight-bearing was observed within a day after surgery (Animated Figure 1). The weight of the osteosynthesis (plate and screws) used in the present study was about 0.1% of the mouse body weight. No postoperative complications (e.g., wound infection, implant failure, bone graft migration, etc.) occurred. No self-injury or injuries caused by cagemates occurred.
When the surgically-induced bone defects were left empty, no significant bone formation was observed with consistent bone non-union. In contrast, when the defects were filled with either an isograft or a coral scaffold, newly-formed bone extending from the proximal and distal bone edges was observed. In addition, whereas bone formation allowed re-establishment of bone continuity in most defects treated with isografts (Figure 8), it was only observed inside the coral scaffold in defects filled with this material. In fact, no bone was observed at a distance greater than 1 mm from the bony edges. Absence of cartilage in all histological analyses results provided evidence of the stability of the achieved osteosynthesis (Figure 9, Figure 10).
Radiographs and microCT analyses provided evidence that bone union did not occur in any animal of the defect-left-empty group, 10 weeks post implantation. The volume of mineralized tissue assessed by microCT analyses was 0.8 &#177; 0.3 mm3 and was representative of the newly formed-bone. In the isograft and coral scaffold groups, bone union was obtained in 4 and 4 animals respectively. The volume of mineralized tissue assessed by microCT analyses was 4.4 &#177; 0.9 mm3 and 8.9 &#177; 0.7 mm3. In these groups, however, because both the isograft and the coral scaffold contained minerals, new bone formation could not be truly distinguished from the remaining implanted material (isograft or coral scaffold). Both the rate of bon union and the volume of mineralized tissue obtained from the isograft group and from the coral scaffold group were significantly (p &#60; 0.001) higher than those obtained from the defect-left-empty group.
<img alt="Figure 1" src="/files/ftp_upload/52940/52940fig1.jpg" /> 
Figure 1: Surgical Exposure for Creation of the Femoral Segmental Defect. A 15 - 17-mm longitudinal skin incision, extending from the hip joint to the stifle joint, was made over the anterolateral aspect of the femur. The fascia lata was incised; the vastus lateralis muscle and the biceps femoris muscle were split to expose the full length of the femoral diaphysis. <a href="https://www.jove.com/files/ftp_upload/52940/52940fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52940/52940fig2.jpg" /> 
Figure 2: Plate Positioning and Proximal Screw Placement. The plate was applied on the anterior femoral side. The most proximal hole of the plate was drilled; the first screw was inserted and, then, locked. <a href="https://www.jove.com/files/ftp_upload/52940/52940fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52940/52940fig3.jpg" /> 
Figure 3: Distal Screw Placement. The most distal hole of the plate was drilled and the screw was inserted and locked. (Reprinted with permission from Tissue Eng Part C, 2013, 19(4), 271-280) <a href="https://www.jove.com/files/ftp_upload/52940/52940fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52940/52940fig4.jpg" /> 
Figure 4: Gigli Saw Positioning. The two other outer screws were inserted but not locked and the wire of the 0.22 mm Gigli saws was tied closely around the bone in a medio-lateral orientation. <a href="https://www.jove.com/files/ftp_upload/52940/52940fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52940/52940fig5.jpg" /> 
Figure 5: Jig Positioning. The jig was inserted on the stem of the two last screws and applied above the plate and the wire of the saw was then inserted in the slots of the jig. (Reprinted with permission from Tissue Eng Part C, 2013, 19(4), 271-280) <a href="https://www.jove.com/files/ftp_upload/52940/52940fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52940/52940fig6.jpg" /> 
Figure 6: Ostectomy. Ostectomy was performed and the Gigli saw was withdrawn. (Reprinted with permission from Tissue Eng Part C, 2013, 19(4), 271-280) <a href="https://www.jove.com/files/ftp_upload/52940/52940fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52940/52940fig7.jpg" /> 
Figure 7: Inner Screws Locking. The jig was removed and the two last screws locked. The segmental defects were then either left empty or filled with the materials tested. (Reprinted with permission from Tissue Eng Part C, 2013, 19(4), 271-280) <a href="https://www.jove.com/files/ftp_upload/52940/52940fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/52940/52940fig8.jpg" /> 
Figure 8: Representative Postoperative Radiographs and Sagittal &#956;CT Reconstruction of the Femoral Bone of Mice. Femoral bone with the respective defect either left empty (A-E), or filled with massive syngenic bone graft (F-J), or filled with massive Acropora coral scaffolds (K-O); immediately after surgery (A, F, K), 4 weeks after surgery (B, G, L), 6 weeks after surgery (C, H, M), and 10 weeks after surgery (D, E, I, J, N, O) (plate length = 10 mm). (Reprinted with permission from Tissue Eng Part C, 2013, 19(4), 271-280) <a href="https://www.jove.com/files/ftp_upload/52940/52940fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/52940/52940fig9.jpg" /> 
Figure 9: Representative Radiograph, &#956;CT Reconstruction and Histology of a Defect Filled with the Coral Scaffold Tested in the Present Study. A large amount of newly formed bone was observed in-between the surrounding bony edges and the coral scaffold; in contrast, little bone was present inside the scaffold. Stains: Stevenel Blue and von Gieson picrofuchsin. Under these conditions, bone, cells, and coral stained red, blue, and brown, respectively. Scale bar = 500 &#956;m. ACS = Acropora coral scaffold; BN = bone. (Reprinted with permission from Tissue Eng Part C, 2013, 19(4), 271-280) <a href="https://www.jove.com/files/ftp_upload/52940/52940fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/52940/52940fig10.jpg" /> 
Figure 10: Representative Histology of a Defect Left Empty (A), Filled with Massive Syngenic Bone Graft (B), and Filled with Coral Scaffold (C). In the defect left empty, rounding of the bony edges with medullary canal filling and abundant fibrous tissue deep into the defect were observed. In the defect filled with massive syngenic bone graft, bone continuity was observed between the graft and the surrounding bony edges; bone marrow was present throughout the original cavity. In the defect filled with coral scaffold, newly formed bone was observed between the surrounding bony edges and the coral scaffold, but little bone was present inside the scaffold. Stains: Stevenel Blue and von Gieson picrofuchsin. Under these conditions, bone, cells, and coral stained red, blue, and brown, respectively. Scale bar = 500 mm. ACS, coral scaffold; BN, bone; BM, bone marrow; FT, fibrous tissue.(Reprinted with permission from Tissue Eng Part C, 2013, 19(4), 271-280) <a href="https://www.jove.com/files/ftp_upload/52940/52940fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Animated Figure 11" src="/files/ftp_upload/52940/52940animfig1.jpg" /> 
Animated/video Figure 1: Representative video of the gait of a mouse one day postoperative. Full weight bearing was observed. <a href="https://www.jove.com/files/ftp_upload/52940/Animated_Figure1.MOV" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis at JoVE.com

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

establishment-segmental-femoral-critical-size-defect-model-mice

Universidad San Sebastian

Research

JoVE Journal

Medicine

10.0K Views.  Université Paris Diderot. Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Video: Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

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 ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found at the end of all long bones in children that is responsible for longitudinal bone growth. Once damaged, cartilage tissue within the growth plate can undergo premature ossification and lead to unwanted bony repair tissue, which forms a "bony bar." In some cases, this bony bar can result in bone growth deformities, such as angular deformities, or it can completely halt longitudinal bone growth. There is currently no clinical treatment that can fully repair an injured growth plate. Using an animal model of growth plate injury to better understand the mechanisms underlying bony bar formation and to identify ways to inhibit it is a great opportunity to develop better treatments for growth plate injuries. This protocol describes how to disrupt the rat proximal tibial growth plate using a drill-hole defect. This small animal model reliably produces a bony bar and can result in growth deformities similar to those seen in children. This model allows for investigation into the molecular mechanisms of bony bar formation and serves as a means to test potential treatment options for growth plate injuries.

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

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 ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...