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>

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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...

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&#34;,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 &#38; 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&#38;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 &#34; 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...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.5K Views.  University of Wisconsin-Madison. 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.

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 ...

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 ...

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) ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

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

Summary

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Chapters in this video

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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

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

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

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

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

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid