This work was supported by an NIH Equipment Grant 1S10OD023676-01 with additional support provided through the University of Wisconsin&#8217;s Departments of Orthopedics and Rehabilitation and School of Medicine and Public Health. We wish to acknowledge the UW&#8217;s Carbone Cancer Center Support Grant P30 CA014520 and use of their Small Animal Imaging Facility, as well as NIH Training Grant 5T35OD011078-08 for support of H. Martin. We also thank Michael and Mary Sue Shannon for their support of the Musculoskeletal Regeneration Partnership.

The rats used in this study received daily care in accordance with the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition16. The Institutional Animal Care and Use Committee at the University of Wisconsin-Madison evaluated and approved this experimental protocol before the project began.
1. Animals
<ol>
	<li>Use the outbred Sprague-Dawley male rats weighing approximately 350 g.</li>
</ol>
2. Preparation of Bone Morphogenetic Protein-2 (rhBMP-2) soaked sponge scaffolds
NOTE: Scaffold preparation should occur just before implantation in the femur (see Step 6.14).
<ol>
	<li>Follow manufacturer instructions for the use of an established rhBMP-2 bone graft kit containing a collagen sponge, lyophilized rhBMP-2, and sterile water for reconstitution17. Maintaining sterility, reconstitute the rhBMP-2 with the sterile water to a concentration of 1.5 mg/mL.</li>
	<li>Using sterile scissors and a sterile ruler, trim the rhBMP-2 soaked collagen sponge to reshape to fit a 5 mm x 3 mm x 3 mm defect.</li>
	<li>Using a syringe, distribute the rhBMP-2 solution evenly over the collagen sponge so that it is absorbed.</li>
</ol>
3. Preparation of custom external fixation device
NOTE: See Figure 1A for the more complete listing of dimensions.
<ol>
	<li>Cut aluminum sheet stock (type 6061, 0.088&#8221; thickness) to two pieces (1.4&#8221; x 6&#8221;) using a jigsaw or other appropriate tool.</li>
	<li>Mount one piece in the milling machine and, using a 1/8&#8221; 90&#176;-point carbide drill mill, cut four &#39;V&#39; grooves (0.035&#8221; deep) lengthwise. Leave the other piece free of cuts.</li>
	<li>Cut individual plates of 0.3&#8221; width from the two pieces (Figure 1B). Measure and drill screw holes for 4-40 thread. Tap plate with &#8216;V&#8217; grooves with the 4-40 thread. Drill the plate without grooves for a #4 screw body drill.</li>
	<li>Sand both pieces to round corners and reduce weight (Figure 1C).</li>
	<li>Screw pieces together with 4-40, 18-8 stainless steel button head cap screws (0.25&#8221;) so that grooves are flush against the plain plate (Figure 1D)</li>
</ol>
4. Anesthetic procedure and analgesia
<ol>
	<li>Induce anesthesia by placing the rat in induction chamber delivering 4 L O2/min with 4% isoflurane. 
	CAUTION: Research personnel must avoid inhalation of anesthetic gas and maintain proper hood and ventilation in the laboratory.</li>
	<li>Remove the rat from the chamber after the rat loses righting reflex, attach a nose cone and place at the maintenance dose of anesthesia through the nose (O2 delivery rate to 2-3 L/min and 0.8% isoflurane).</li>
	<li>Place the rat on the heating pad or under the warming light to prevent hypothermia.</li>
	<li>Confirm the adequate depth of anesthesia by pinching the toe or testing the palpebral reflex.</li>
	<li>Apply lubrication to eyes to prevent drying out of the cornea.</li>
	<li>Deliver a subcutaneous injection of extended-release buprenorphine (1 mg/kg) on the trunk/dorsum of the rat, far from the surgical site, to provide analgesia for up to 3 days following surgery.</li>
</ol>
5. Aseptic preparation and antibiotic preventatives
<ol>
	<li>Shave area around hindleg using the 13th rib, the foot, the dorsal midline, and the ventral midline as margins.</li>
	<li>Scrub shaved area using sterile 2 x 2 gauze soaked with 10% povidone-iodine followed by 70% EtOH (4 times each, alternating).</li>
	<li>Administer an intramuscular injection of cefazolin (20 mg/kg) into the operative quadriceps.</li>
	<li>Administer enrofloxacin (0.25 mg/ml) in drinking water for 7 days postoperatively for continued antibiotic protection.</li>
	<li>Place rats on medicated feed (e.g., Uniprim) for the duration of the study to prevent pin tract infections.</li>
	<li>Apply double antibiotic ointment to the skin-pin interface once daily for 3 days postoperatively. 
	NOTE: Avoid any external fixation pin or clamp loosening which can contribute to the development of an infection.</li>
</ol>
6. Surgical procedure
NOTE: Make a concerted to effort to maintain a sterile field and workspace and follow sterile technique throughout the entirety of the case.
<ol>
	<li>Extend shaved leg through fenestrated, clear sticky drape and cover surgical bench in sterile towels to create a sterile field.</li>
	<li>Palpate the femur and use a #15 blade to create an anterolateral incision through the skin extending from the patella to the greater trochanter at the proximal femur.</li>
	<li>Carefully incise the lateral leg fascia along the intermuscular septum to separate the vastus lateralis muscle of the quadriceps anteriorly from the hamstrings posteriorly until the lateral femur is exposed. Preserve the abductor gluteal tendon insertion on the greater trochanter.</li>
	<li>Perform a careful, atraumatic circumferential soft tissue dissection and expose the femur at its mid-diaphysis starting on the lateral surface. To do this, use a #15 blade to gently cut the muscle away from the underlying bone by keeping the blade parallel against the contour of the bone surface. Use a periosteal elevator to lift the muscle away from the exposed bone as it is dissected and proceed around the femoral shaft until 7-10 mm of central diaphysis has been cleared of soft tissue on all sides to prepare for ostectomy. 
	NOTE: Avoid injury to the medial femoral neurovascular bundle.</li>
	<li>Insert four 1.0 mm Kirschner (k) wires: 2 proximal and 2 distal in the femur perpendicular to the lateral femur, directed straight lateral to medial. Ensure all pins engage both cortices (bicortical) for adequate stability (Figure 2A).</li>
	<li>Start with the distal-most pin first, just at the level of the lateral epicondyle. Place jig flush to the lateral distal femur and insert a 1.0 threaded tip k-wire.</li>
	<li>Maintaining the position of the jig on the bone, identify where the most proximal pin will enter the bone based on the jig holes. Once the position is determined, carefully incise parallel to the fibers of the gluteal tendon as needed to create a small gap in the tissue for the proximal pin to pass through, thus minimizing iatrogenic damage to the tendon. Drill a 1.0 mm non-threaded k-wire in this gap, again ensuring the pin engages both cortices (Figure 2B).</li>
	<li>Maintain the jig&#8217;s position in contact with the bone and drill two 1.0 mm threaded k-wires, one on either side of the future defect site. Ensure pins engage both cortices (Figure 2C).</li>
	<li>Place the external fixator bar level 1 cm above skin and screw tightly, locking the bar in place. Clip the excess pin lengths (Figure 2D).</li>
	<li>Prepare for the ostectomy (defect creation) by placing a small, curved retractor around the anterior and posterior femur to protect the surrounding soft tissue, muscle, and neurovascular bundle. Utilizing a ~5 mm sagittal oscillating saw blade, very cautiously create a 5 mm segmental defect through the mid-diaphysis. Apply a light, even pressure with the saw to avoid unnecessary fracture (Figure 2E).</li>
	<li>Apply small amounts of irrigation (room temperature 0.9% sterile normal saline (NS)) as needed while creating defect to avoid thermal necrosis of the bone.</li>
	<li>Flush the wound using 10 mL of NS after creating the defect.</li>
	<li>Administer 0.1 mL of a 0.25% bupivacaine with epinephrine (1:200,000) to the wound as an analgesic and vasoconstrictor.</li>
	<li>Insert the scaffold (5 mm x 3 mm x 3 mm) of collagen sponge or rhBMP-2 soaked sponge (from Step 2) into the defect. Each scaffold should be sized appropriately to span the length and volume of the defect, helping the sponge stay in position. 
	NOTE: At this point, mRNA complexes may be prepared and injected as outlined in Steps 7.1-7.3 below if performing bioluminescence imaging.</li>
	<li>Close the muscle plane using the simple interrupted pattern with 4-0 absorbable suture. Close the skin layer using a running subcuticular pattern with 4-0 absorbable suture and skin glue to close gaps around the protruding pins.</li>
	<li>Remove the rat from the nose cone, remaining on the heating pad, and monitor continuously until the rat is able to consistently maintain an upright posture. At this point, place in a clean cage to recover.</li>
</ol>
7. Preparation of complexed mRNA and bioluminescence imaging
NOTE: Transfection with mRNA complexes should be performed during surgery 1 day before luminescence imaging. Use sterile techniques when handling mRNA.
<ol>
	<li>Mix 10 &#181;L of mRNA encoding for Gaussia luciferase (stock concentration of 1 &#181;g/&#181;L) with 30 &#181;L of the lipidic transfecting agent.</li>
	<li>Allow for the mRNA-lipid complexes to form by incubating for at least 5 min at room temperature. The lipidic transfecting agent will condense the mRNA molecules, stabilizing them and enhancing transfection efficiency. 
	NOTE: If the complexes are not used immediately, store them in ice for a maximum of 1 h.</li>
	<li>Using a 20 &#181;L pipette equipped with filtered tips, inject half of the volume of mRNA complexes to the distal and proximal ends of the defect, respectively.</li>
	<li>The following day, 3 min before imaging, anesthetize the rat using inhaled isoflurane as previously described in Step 4.1.</li>
	<li>Position the rat in an in vivo imaging chamber equipped with a nose cone delivering maintenance isoflurane (0.8% isoflurane, O2 delivery rate of 2-3 L/min).</li>
	<li>Inject coelenterazine resuspended in saline at a dose of 4 mg/kg body weight in the proximity of the defect.</li>
	<li>Acquire bioluminescence images with the in vivo imaging system (IVIS) according to the manufacturer&#8217;s instructions18.</li>
</ol>
8. Imaging Protocol
<ol>
	<li>After calibrating the plain radiographic machine, an X-ray system19, anesthetize rat using inhaled isoflurane as previously described (see Step 4.1) and position the rat in a nose cone with inhaled isoflurane (0.8% isoflurane, O2 delivery rate of 2-3 L/min) for an anteroposterior (AP) femur radiograph.
	<ol>
		<li>While the rat is in sternal recumbency, advance the surgical hindlimb forward, flexing at the hip and stifle joint. Flex the stifle joint to approximately 90&#176;. Tape the paw plantar side down, close to the body wall. Position the tibia forward from the femur to eliminate the possibility of superimposing the bones. To provide slight abduction of the hip, place a translucent sponge (approximately 15 mm thick) in the groin region. Then obtain an anterior-posterior (cranial-caudal) image of the femur.</li>
	</ol>
	</li>
	<li>Repeat this AP femur radiographic view immediately following surgery, 4 weeks, and 12 weeks. Use tape and gauze to appropriately position the animal&#8217;s extremity for quality and consistent imaging.</li>
	<li>Remove the rat from the nose cone and monitor continuously until the rat is able to consistently maintain an upright posture. Then, place back into the cage.</li>
</ol>
9. Histological Procedure
<ol>
	<li>Euthanize rats in a chamber with inhaled CO2 according to AVMA ethical standards16.</li>
	<li>Following euthanasia, shave the hindlimb, remove the skin from the operative extremity and disarticulate femur at the hip. Carefully remove all soft tissue from the operative femur (including all muscles, tendons, and ligaments). Leave only a thin layer of muscle surrounding the defect site to protect the healing region from inadvertent damage during dissection.</li>
	<li>Place the femur in 10% Neutral Buffered Formalin at room temperature for 3-4 days to allow for fixation. Keep a 15:1 formalin to tissue volume ratio. Change the solution once halfway through the fixation process.</li>
	<li>Decalcify the femur in a 15% Ethylenediaminetetraacetic acid (EDTA) pH 6.5 solution for 3-4 weeks. Collect serial radiographs to determine decalcification endpoint.</li>
	<li>Bisect the femur longitudinally with a cut from anterior to posterior in the mid-sagittal plane. Submit tissue for standard paraffin embedding and hematoxylin and eosin (H&#38;E) staining.</li>
	<li>Send H&#38;E slides to a pathologist for histological assessment.</li>
</ol>

<ol>
	<li>Filipowska, J., Tomaszewski, K. A., Nied&#378;wiedzki, &. #. 3. 2. 1. ;., Walocha, J. A., Nied&#378;wiedzki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+vasculature+in+bone+development,+regeneration+and+proper+systemic+functioning.">The role of vasculature in bone development, regeneration and proper systemic functioning.</a> Angiogenesis. 20 (3), 291-302 (2017).</li><li>Charalambous, C. P., Akimau, P., Wilkes, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+monolateral-ring+fixator+for+bone+transport+in+post-traumatic+femoral+segmental+defect:+A+technical+note.">Hybrid monolateral-ring fixator for bone transport in post-traumatic femoral segmental defect: A technical note.</a> Archives of Orthopaedic and Trauma Surgery. 129 (2), 225-226 (2009).</li><li>Xing, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+bilateral+femoral+large+segmental+bone+defect+mouse+model+potentially+applicable+to+basic+research+in+bone+tissue+engineering.">Establishment of a bilateral femoral large segmental bone defect mouse model potentially applicable to basic research in bone tissue engineering.</a> The Journal of Surgical Research. 192 (2), 454-463 (2014).</li><li>Chadayammuri, V., Hake, M., Mauffrey, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+strategies+for+the+management+of+long+bone+infection:+A+review+of+the+Masquelet+technique.">Innovative strategies for the management of long bone infection: A review of the Masquelet technique.</a> Patient Safety in Surgery. 9 (32), (2015).</li><li>Koettstorfer, J., Hofbauer, M., Wozasek, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+limb+salvage+using+the+two-staged+technique+with+internal+fixation+after+osteodistraction+in+an+effort+to+treat+large+segmental+bone+defects+in+the+lower+extremity.">Successful limb salvage using the two-staged technique with internal fixation after osteodistraction in an effort to treat large segmental bone defects in the lower extremity.</a> Archives of Orthopaedic and Trauma Surgery. 132 (19), 1399-1405 (2012).</li><li>Fragomen, A. T., Rozbruch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanics+of+external+fixation.">The mechanics of external fixation.</a> The Musculoskeletal Journal of Hospital for Special Surgery. 3 (1), 13-29 (2007).</li><li>O&#8217;Toole, R. V., 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=A+prospective+randomized+trial+to+assess+fixation+strategies+for+severe+open+tibia+fractures:+Modern+ring+external+fixators+versus+internal+fixation+(FIXIT+Study).">A prospective randomized trial to assess fixation strategies for severe open tibia fractures: Modern ring external fixators versus internal fixation (FIXIT Study).</a> Journal of Orthopaedic Trauma. 31, S10-S17 (2017).</li><li>F&#252;rmetz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+transport+for+limb+reconstruction+following+severe+tibial+fractures.">Bone transport for limb reconstruction following severe tibial fractures.</a> Orthopedic Reviews. 8 (1), 6384 (2016).</li><li>Dohin, B., Kohler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Masquelet&#8217;s+procedure+and+bone+morphogenetic+protein+in+congenital+pseudarthrosis+of+the+tibia+in+children:+A+case+series+and+meta-analysis.">Masquelet&#8217;s procedure and bone morphogenetic protein in congenital pseudarthrosis of the tibia in children: A case series and meta-analysis.</a> Journal of Children's Orthopaedics. 6 (4), 297-306 (2012).</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> Nature Reviews Rheumatology. 11, 45-54 (2015).</li><li>Pascher, 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=Gene+delivery+to+cartilage+defects+using+coagulated+bone+marrow+aspirate.">Gene delivery to cartilage defects using coagulated bone marrow aspirate.</a> Gene Therapy. 11 (2), 133-141 (2004).</li><li>Glatt, V., Matthys, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjustable+stiffness,+external+fixator+for+the+rat+femur+osteotomy+and+segmental+bone+defect+models.">Adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models.</a> Journal of Visualized Experiments. (92), (2014).</li><li>Betz, O. B., 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=Direct+percutaneous+gene+delivery+to+enhance+healing+of+segmental+bone+defects.">Direct percutaneous gene delivery to enhance healing of segmental bone defects.</a> The Journal of Bone and Joint Surgery. 88 (2), 355-365 (2006).</li><li>Fang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+new+bone+formation+by+direct+transfer+of+osteogenic+plasmid+genes.">Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (12), 5753-5758 (1996).</li><li>Kaspar, K., Schell, H., Toben, D., Matziolis, G., Bail, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easily+reproducible+and+biomechanically+standardized+model+to+investigate+bone+healing+in+rats,+using+external+fixation.">An easily reproducible and biomechanically standardized model to investigate bone healing in rats, using external fixation.</a> Biomedizinische Technik. 52 (6), 383-390 (2007).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AVMA+guidelines+for+the+euthanasia+of+animals:+2013+edition.">AVMA guidelines for the euthanasia of animals: 2013 edition.</a> American Veterinary Medical Association. , (2013).</li><li>McKay, W. F., Peckham, S. M., Badura, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+clinical+review+of+recombinant+human+bone+morphogenetic+protein-2+(INFUSE+Bone+Graft).">A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE Bone Graft).</a> International Orthopaedics. 31 (6), 729-734 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Living lmage Software. , (2006).</li><li>Bassett, J. H. D., Van Der Spek, A., Gogakos, A., Williams, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+X-ray+imaging+of+rodent+bone+by+faxitron.">Quantitative X-ray imaging of rodent bone by faxitron.</a> Methods in Molecular Biology. , 499-506 (2012).</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>Lieberman, J. R., 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=The+effect+of+regional+gene+therapy+with+bone+morphogenetic+protein-2-producing+bone-marrow+cells+on+the+repair+of+segmental+femoral+defects+in+rats.">The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats.</a> The Journal of Bone and Joint Surgery. 81 (7), 905-917 (1999).</li><li>Tsuchida, H., Hashimoto, J., Crawford, E., Manske, P., Lou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+allogeneic+mesenchymal+stem+cells+repair+femoral+segmental+defect+in+rats.">Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats.</a> Journal of Orthopaedic Research. 21 (1), 44-53 (2003).</li><li>Jiang, H., 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=Novel+standardized+massive+bone+defect+model+in+rats+employing+an+internal+eight-hole+stainless+steel+plate+for+bone+tissue+engineering.">Novel standardized massive bone defect model in rats employing an internal eight-hole stainless steel plate for bone tissue engineering.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (4), 2162-2171 (2018).</li><li>Baltzer, A. W., 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=Genetic+enhancement+of+fracture+repair:+Healing+of+an+experimental+segmental+defect+by+adenoviral+transfer+of+the+BMP-2+gene.">Genetic enhancement of fracture repair: Healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene.</a> Gene Therapy. 7 (9), 734-739 (2000).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+defect+animal+models+for+testing+efficacy+of+bone+substitute+biomaterials.">Bone defect animal models for testing efficacy of bone substitute biomaterials.</a> Journal of Orthopaedic Translation. 3 (3), 95-104 (2015).</li></ol>

The authors declare no competing financial interests or benefits. There have been no benefits received directly or indirectly by the authors of this article.

Small animal models of orthopedic injuries such as complete bone fractures enable research that explores the mechanisms of osteogenesis and assessing the therapeutic potential of biomaterials20. This study introduces a rat segmental defect model stabilized by a custom external fixator that a lab and biomedical engineering team can readily reproduce for further studies of load-bearing osteosynthetic bone repair.
Previous studies using critical-sized defects in rat models commonly rely on internal fixation plates21,22,23,24. Although either fixation method is clinically acceptable, external fixation has the distinct advantages of causing less soft tissue disruption and blood loss, decreasing the total implanted surface area to minimize opportunities for bacterial colonization, and allowing for postoperative adjustability and staged surgical interventions6. Previous external fixation animal fracture models have used different plate materials, pin securement, and/or bone cutting methods12,13,14. Because the external fixator in this protocol was made in the lab&#8217;s workshop with low-cost aluminum and can be easily refurbished, expenses were minimized. This provides an economical external fixator allows researchers to perform experiments with multiple animal groups without being limited financially. As compared to an internal fixation model, we believe that this system is technically simpler and more reproducible in a small animal model. In our experience with these models, internal fixation is significantly more demanding technically and can require custom-machined implants. To the authors&#8217; knowledge, this overall protocol is unique in its use of a custom-designed jig combined with an adapted metal external fixator design15, as well as its use of an oscillating bone saw to better represent the common clinical scenario of periosteal stripping performed to prepare fracture sites for fixation. A final note on the use of an oscillating saw in this model is that the heat generated and the periosteal disruption that occurs provides a final element of control in forming a non-union model. Our experience has been that with other methods, in these animal models, investigators run the risk of the controls healing.
To ensure surgical success, care should be taken at several critical steps: when incising and performing the circumferential dissection to expose the femur from surrounding muscle, avoid disturbing the sciatic nerve caudally, the femoral vessels medially, and the gluteal tendon proximally. Take care to position the jig apparatus parallel and flush against the flat, anterolateral face of the femur so that all pins are precisely perpendicular to the bone. This will confirm the proper alignment of the external fixator and reduce the likelihood of pin breakage. The order of pin placement found to be most straightforward was distal first, followed by proximal and then both middle pins. This allowed for less disruption of the gluteal tendon by the proximal pin. Finally, it is important that each pin is placed bicortically, penetrating both cortices so that it does not back out of the bone or shift into the medullary cavity.
An x-ray machine capable of high-resolution radiography was used to monitor bone-healing status as qualitative changes can be visualized easily and non-invasively over time. However, consistent leg positioning is crucial for accurate radiographic interpretation as differences in positioning may be misleading. Results from this study are in agreement with previous work demonstrating that a 5 mm femoral defect prevents spontaneous bone healing in normal rats25. Therefore, any healing that is noted with added therapies such as the rhBMP-2 soaked sponge can be definitively attributed to the respective treatment (Figure 3).
Possible concerns for this technique include pin breakage, loosening, and infection. Problems with proximal pin breakage in preliminary testing prompted a switch from threaded to non-threaded k-wire. Non-threaded pins are mechanically stronger but also pose more risk of backing out of the cortex. Particularly in empty bone defects, k-wires may break or be displaced around 8 weeks due to the prolonged cyclic pin loading and lack of healing (similarly to cycling/bending a paperclip). A strict antibiotic regimen was utilized to include an immediate cefazolin injection, daily application of antibiotic ointment at the incision site, 7 days of enrofloxacin added to drinking water, and a medicated feed. This antibiotic protocol, along with the proper surgical techniques outlined above, minimized infection (Figure 5).
One of the additional advantages of using external fixation in an animal model is easy removal for an unobstructed view of the defect and replacement after imaging. This enables more effective in vivo imaging techniques relying on fluorescence or luminescence to assess changes such as the gene or protein expression. For instance, we have shown that cells in the medullary cavity transfected with complexed mRNA encoding for Gaussia luciferase could be visualized with IVIS. Figure 6 illustrates that luminescence signal detection capability is not obstructed by this external fixation approach as it may be with internal plates, screws, or intramedullary nails21,22,23,24. This cost effective and reproducible surgical protocol allows for consistent creation and stabilization of a critical-sized femoral defect that mimics the initial clinical management of these complex fractures. The establishment of a reliable animal model is critical for any experimental treatments intended for eventual clinical use. Our model has demonstrated predictable results and minimal behavioral changes or discomfort in our animals. This model may be used with a variety of biomaterial-based scaffolds in conjunction with the imaging techniques utilized in this paper for future translational testing purposes. It is our hope that in working with this model, researchers will be able to devise novel ways to treat critical bony defects in trauma patients. This could help avoid the morbidity and cost in lengthy treatments currently employed and possibly decrease the number of amputations.

Orthopedic trauma surgery focuses on treating a wide range of complex fractures. Critical diaphyseal segmental bone defects have proven difficult to treat clinically due to the decreased regenerative ability of the surrounding muscle and periosteum as well as the failure of localized angiogenesis1. Modern treatment techniques include operative fixation with bone grafting, delayed bone (Masquelet) grafting, bone transport, fusion, or amputation2,3,4. In most patients who have ambulatory function preserved after their trauma, with well-functioning distal limbs, limb salvage is clearly a better treatment option5. These salvage treatments often require staged surgical interventions over a long treatment course. Some authors have suggested that external fixation is superior as compared to the internal fixation for these applications due to the decreased tissue damage during implantation, decreased implanted surface area, and increased postoperative adjustability of the fixator6. However, a prospective randomized controlled trial is currently underway to help clarify this controversy of internal versus external fixation in severe open fractures of the tibia7. Unfortunately, with either treatment selected, significant complication and failure rates persist8,9. With either treatment method, with respect to the segmental bone loss, the surgeon must contend with segmental diaphyseal defects that present significant challenges. Corrections of segmental defects must maximize bone stabilization and simultaneously enhance the osteogenic process10,11.
Due to the clinical importance, yet the lower volume, of critical-sized diaphyseal segmental defects, an effective, reproducible animal model is necessary to enable research teams to advance treatment techniques and ultimately improve clinical outcomes. Researchers need to study in vivo physiologic healing mechanisms in a mammalian animal model. While such models of external fixation already exist12,13,14,15, we hope to provide a more reliable method for non-unions in the untreated animals, decrease costs through the choice of affordable fixator materials, and outline a straightforward surgical protocol for the easy application to future studies. The primary goal of this protocol is to establish a reliable and reproducible model of a critical diaphyseal defect in rats. The procedure was evaluated by assessing the stabilization and bone healing in rat femurs over 12 weeks. The secondary goals included: making an affordable model as a cost effective as possible, simplifying the surgical approach and stabilization, and ensuring ethical care of the animals. The authors and research team conducted preliminary experiments with a range of different biomaterials and potential regenerative therapies to improve healing in this segmental defect.

<table><tbody><tr><td>0.9% Sterile Saline</td><td>Baxter</td><td>2F7124</td><td>Used for irrigating wound and rehydration</td></tr><tr><td>10% Iodine/Povidone</td><td>Carefusion</td><td>1215016</td><td>Used to prep skin</td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>89370094</td><td>Used as fixative</td></tr><tr><td>1mm non-threaded kirschner wire</td><td>DePuy Synthes</td><td>VW1003.15</td><td>Sterilized, used for the most proximal pin</td></tr><tr><td>1mm threaded kirschner wire</td><td>DePuy Synthes</td><td>VW1005.15</td><td>Sterilized, used for the 3 most distal pin slots</td></tr><tr><td>2x2 gauze</td><td>Covidien</td><td>4006130</td><td>Sterilized, used to prep skin and absorb blood</td></tr><tr><td>4-0 Vicryl Suture</td><td>Ethicon</td><td>4015304</td><td>Used to close muscle and skin layers</td></tr><tr><td>4-40 x 0.25&quot;,18-8 stainless steel button head cap screws</td><td>Generic</td><td></td><td>External fixator assembly</td></tr><tr><td>4200 Cordless Driver</td><td>Stryker</td><td>OR-S-4200</td><td>Used to drill kirschner wires</td></tr><tr><td>4x4 gauze</td><td>Covidien</td><td>1219158</td><td>Sterilized, used to absorb blood</td></tr><tr><td>70 % Ethanol</td><td></td><td></td><td>Used to prep skin</td></tr><tr><td>Baytril</td><td>Bayer Healthcare LLC, Animal health division</td><td>312.10010.3</td><td>Added to water as an antibiotic</td></tr><tr><td>Cefazolin</td><td>Hikma Pharmaceuticals</td><td>8917156</td><td>Pre-op antibiotic</td></tr><tr><td>CleanCap Gaussia Luciferase mRNA (5moU)</td><td>TriLink Biotechnologies</td><td>L-7205</td><td>Modified mRNA encoding for Gaussia Luciferase, keep on ice during use</td></tr><tr><td>Coelenterazine native</td><td>NanoLight Technology</td><td>303</td><td>Substrate for Guassia Luciferase, used to assess luciferase activity in vivo</td></tr><tr><td>Double antibiotic ointment</td><td>Johnson &amp;amp; Johnson consumer Inc</td><td>8975432</td><td>Applied to pin sites post-op as wound care</td></tr><tr><td>Dual Cut Microblade</td><td>Stryker</td><td>5400-003-410</td><td>Used to create 5mm defect in femur</td></tr><tr><td>Ethylenediamine Tetraacetic Acid (EDTA)</td><td>Fisher</td><td>BP120-500</td><td>Used to decalcify bone to prep for histology</td></tr><tr><td>Extended Release Buprenorphine</td><td>ZooPharm</td><td></td><td>Used as 3 day pain relief</td></tr><tr><td>Fenestrated drapes</td><td>3M</td><td>1204025</td><td>Used to establish sterile field</td></tr><tr><td>Handpiece cord for TPS</td><td>Stryker</td><td>OR-S-5100-4N</td><td>Used to create 5mm defect in femur</td></tr><tr><td>Heating pad</td><td>K&amp;amp;H Pet Products</td><td>121239</td><td>Rat body temperature maintenance</td></tr><tr><td>Hexagonal head screwdriver</td><td>Wiha</td><td>263/1/16 &quot; X 50</td><td>External fixator tightening</td></tr><tr><td>Induction chamber</td><td>Generic</td><td></td><td>Anesthesia for rats</td></tr><tr><td>Infuse collagen sponge with recombinant human Bone Morphogenic Protein-2</td><td>Medtronic</td><td>7510200</td><td>Clinically relevant treatment used as positive control</td></tr><tr><td>Isoflurane</td><td>Clipper</td><td>10250</td><td>Anesthesia for rats</td></tr><tr><td>IVIS</td><td>Perkin Elmer</td><td>124262</td><td>Bioluminescence imaging modality</td></tr><tr><td>Jig</td><td>Custom</td><td></td><td>Used to place bicortical pins</td></tr><tr><td>Lipofectamine MessengerMAX</td><td>Fisher Scientific</td><td>LMRNA003</td><td>mRNA complexing agent that enables mRNA delivery</td></tr><tr><td>Sensorcaine-MPF (Bupivicane (0.25%) and Epinephrine (1:200,000))</td><td>APP Pharmaceuticals, LLC</td><td>NDC 63323-468-37</td><td>Applied to surgical site for pain relief and vasoconstriction</td></tr><tr><td>Sterile water</td><td>Hospira</td><td>8904653</td><td>Used as solvent for cefazolin powder</td></tr><tr><td>Titanium external fixator plates</td><td>Custom</td><td></td><td>Prepared in house with scrap titanium and milling machine</td></tr><tr><td>Total Performance System (TPS) Console</td><td>Stryker</td><td>OR-S-5100-1</td><td>Used to create 5mm defect in femur</td></tr><tr><td>TPS MicroSaggital Saw</td><td>Stryker</td><td>OR-S-5100-34</td><td>Used to create 5mm defect in femur</td></tr><tr><td>Ultrafocus Faxitron with DXA</td><td>Faxitron</td><td></td><td>High resolution radiographic imaging modality</td></tr><tr><td>Uniprim rat diet</td><td>Envigo</td><td>TD.06596</td><td>Medicated rat diet</td></tr><tr><td>Universal Handswitch for TPS</td><td>Stryker</td><td>OR-S-5100-9</td><td>Used to create 5mm defect in femur</td></tr><tr><td>Vetbond Tissue Adhesive</td><td>3M</td><td>1469</td><td>Skin closure</td></tr></tbody></table>

a reliable and reproducible critical-sized segmental femoral defect model in rats stabilized with a custom external fixator

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

Surgeries were performed in approximately one hour by one surgeon with the help of one assistant. After surgical optimization, intra- and postoperative complications were greatly minimized and use of the jig apparatus ensured consistent size (5 x 3 x 3 mm) and localization of femoral defects. Rats were ambulatory immediately following recovery from anesthesia and did not appear to have any altered behavioral patterns; their gait was not antalgic, and they did not appear to be disturbed by the external fixator.
Non-threaded k-wires were chosen for the most proximal pin (Figure 2B), as the proximal pin had the highest risk of breaking when threaded wires were used. In some instances, particularly in control animals without rhBMP-2 or scaffolds whose defects showed no evidence of healing/bone formation, one or more k-wire tips broke after about 8 weeks as seen in the sponge only control radiograph of the excised femur (Figure 3).
Radiographs and histology (H&#38;E stain) were analyzed to assess levels of bone healing. Negative control defects containing only a collagen sponge showed no evidence of bridging osteogenesis between the proximal and distal bone edges (Figure 3, Figure 4). A small amount of new bone remodeling can be seen directly adjacent to the cut femur edge; the defect itself shows a lack of bony material, the presence of cartilage, and some residual hematoma (Figure 4). Defects containing rhBMP-2 soaked sponge demonstrated significant bone healing as early as 4 weeks after surgery, as shown by the radiopaque callous bridging across the defect in Figure 3. By 12 weeks, significant new mineral deposition (Figure 4, NB: new bone, PC: periosteal callous) has formed throughout the defect. Significant new periosteal bone can be seen in the callous extending from the cut femur edge, and spicules of woven and lamellar bone have developed throughout the defect. Cartilage deposition is not seen (Figure 4).
Histology (H&#38;E stain) was also performed for both an uninfected control and an example of an infected femur (Figure 5). The infected femur is significantly enlarged, showing signs of an endosteal reaction infiltrating the bone cortex. Arrows indicate areas of osteoclast-mediated pathologic bone resorption. The uninfected femur cortex remains compact and with a clearly delineated lamellar cortex. Antibiotic dosing was optimized to include maximal coverage postoperatively. While infection around the defect site can occur, continued administration of antibiotics topically around pin sites and in water and diet proved successful in minimizing postoperative infection.
Further imaging using In Vivo Imaging System (IVIS) illustrates the ability of bioluminescent cells to be visualized within the defect after implantation of the external fixator (Figure 6). The external plate can be easily removed for imaging and replaced upon completion. Cells in the medullary cavity luminesce after transfection with complexed mRNA encoding for Gaussia luciferase. The highest level of luminescence is focused at the site of the femoral defect and the signal is not obstructed by the fixation device pins. This is promising for future studies relying on bioluminescence or fluorescence to measure biological changes such as a gene or protein expression during the healing process.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59206/59206fig1.jpg" /> 
Figure 1: External fixator fabrication. A: CAD schematic of the assembled external fixator with annotated dimensions for proper fabrication. Each fixator is composed of two aluminum plates held together by two screws. B: Plates are cut from 1.4&#8221; x 6&#8221; aluminum sheets with &#8216;V&#8217; grooves cut into the bottom sheet. C: Screw holes are drilled into the plates (threaded in the plate with &#8216;V&#8217; grooves) and all edges and corners are sanded to round and reduce weight. D: Assembled external fixator is tightened with screws (4-40 x 0.25&#8221;, 18-8 stainless steel button head cap) once pins are in place in the &#8216;V&#8217; grooves on the insides of the aluminum plates. The left pin is non-threaded and is most proximal on the femur. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59206/59206fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59206/59206fig2.jpg" /> 
Figure 2: Schematic of pin placement, fixator placement, and defect creation. A: The distal pin (1.0 mm threaded k-wire) is placed at the epicondyle metaphyseal region using the jig (blue rectangle) to guide proper pin insertion. The jig is placed onto the anterolateral femoral surface. B: The proximal pin (1.0 mm non-threaded k-wire) is placed using the jig after making a small incision in the gluteal tendon. C: The middle pins (1.0 mm threaded k-wire) are inserted using the jig. D: The jig is removed and the 2 plates are attached to the pins using the 2 screws to secure the plates. The plates are tightened 1 cm above skin level to avoid pressure on the skin. E: A sagittal oscillating saw is used to create a 5 mm defect between the two middle pins. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59206/59206fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59206/59206fig3.jpg" /> 
Figure 3: Representative high-resolution radiographs show bone healing with rhBMP-2 treatment. Images for the negative control collagen sponge and the rhBMP-2 soaked sponge groups are shown at 0, 4, and 12 weeks postoperatively. The rhBMP-2 treatment group exhibits significant healing after 4 weeks with callous spanning the defect. The negative control femur ends do not heal with bridging bone and the defect remains a non-union. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59206/59206fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59206/59206fig4.jpg" /> 
Figure 4: Significant new bone formation is seen with rhBMP-2 treatment. Representative 4x magnified H&#38;E histological images for the negative control collagen sponge and the rhBMP-2 soaked sponge groups both at the cut femur edge and within the defect. New bone formed around the control femur edge, but significant extensions of both new trabecular bone as well as the periosteal callous project from the treated femur. No bony material is seen within the control defect, while significant bone formation can be observed throughout the rhBMP-2 treated defect. NB: new bone, F: femur, C: cartilage, H: hemorrhage, PC: periosteal callous. Scale bar: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59206/59206fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59206/59206fig5.jpg" /> 
Figure 5: Infected femur exhibits hypertrophy and inflammatory cell markers. H&#38;E histological images of an uninfected femur compared to an infected femur, in full view and at 4x magnification of boxed locations. The uninfected femoral cortex remains organized and delineated, with little sign of inflammation. The infected femur enlarges greatly, as seen in full view, and the cortex is broken up by areas of resorption and necrosis (purple cell clusters indicated by black arrows). F: femur. Scale bar: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59206/59206fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59206/59206fig6.jpg" /> 
Figure 6: Gaussia luciferase signal detected in the defect. Luminescence of cells transfected with Gaussia luciferase mRNA is imaged with IVIS after external plate removal. Red indicates the highest luminescence intensity at the site of the femoral defect. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59206/59206fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator at JoVE.com

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

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

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

a-reliable-reproducible-critical-sized-segmental-femoral-defect-model

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.6K Views.  University of Wisconsin-Madison. Dit chirurgische protocol maakt gebruik van een consistent en reproduceerbaar extern vast segmentale defectmodel om de genezingsmechanismen van bot en het gebruik van potentiële regeneratieve therapieën te evalueren. We hebben een uitdagende operatie gestroomlijnd voor het creëren en stabiliseren van een segmentaal botdefectmodel dat toegankelijk en kosteneffectief is en tegelijkertijd geschikt blijft voor longitudinale genezingsstudies. Deze techniek is met name te vertalen naar de behand...

Video: A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

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 ...

Medicine

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing.

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

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 ...

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

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

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.

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 and Evaluation of a Rat Femoral Box: Cavity Defect Model

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

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

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

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

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

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

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