Name: athymic rat model for evaluation of engineered anterior cruciate ligament grafts
Uploaded: 2015-03-26T15:00:00.000+00:00
Duration: 10 min 32 s
Description: Watch this Scientific Journal Video about Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts at JoVE.com

The authors would like to thank Gabriel Arom and Michael Yeranosian for their technical contributions to earlier iterations of this project. This project was funded by the OREF Clinician Scientist Training Grant (NL), H H Lee Surgical Research Grant (NL), Veterans Administration BLR&#38;D Merit Review 1 I01 BX00012601 (DM) and Musculoskeletal Transplantation Foundation Young Investigator Award (FP).

NOTE: All animal surgeries were approved by the local veterinary staff and animal use committee prior to commencing the experiments.
1. Preparation of Electrospun Polycaprolactone Scaffolds
<ol>
	<li>Weigh and dissolve medical grade ester terminated poly (&#949;-caprolactone) in granule form in 1,1,1,3,3,3-hexafluoro-2-propanol to create a 10% w/w solution of the PCL polymer. Let the solution stir using a stir plate for at least 3 hr to ensure a homogenous solution.</li>
	<li>Electrospin the PCL solution to create a cuff of highly aligned PCL fibers for scaffold fabrication.
	<ol>
		<li>Prepare the electrospinning setup in a chemical fume hood with the fan on at all times. This consists of a large acrylic box that will serve as an isolated vacuum medium for the electrospinning process and has entry points for the source PCL solution driven by a voltage source, the motor-powered collecting mandrel, and a vacuum port. Clean the acrylic box thoroughly with ethanol and cover all surfaces with Parafilm sheets to remove any impurities that may compromise the quality of the electrospun product.</li>
		<li>Load approximately 3 ml of the above solution into a 10 ml syringe with a blunt ended 18 G, 1 &#189; inch needle. Remove any air bubbles by pushing the syringe up. Lock solution into a programmable syringe pump. Insert the needle through a small hole into the acrylic box while leaving about &#189; inch of the needle outside of the acrylic box for attachment of the voltage source wire.</li>
		<li>Use a 30 mm rotating lathe mandrel as the collector for the highly aligned PCL fibers; cover the mandrel tightly with a thin strip of aluminum foil. Lock the mandrel into the motor on the opposite side of the box about 15 cm away from the syringe needle.</li>
		<li>Insert the plastic hose into the vacuum port and connect to the fume hood vacuum source. Turn on the vacuum source and cover the acrylic box with a lid.</li>
		<li>Set the infusion rate of the programmable syringe pump to 2.5 ml/hr. Turn on the motor to operate the mandrel at 3,450 rpm and attach the positive lead of the voltage source to the needle tip outside of the box using an alligator clip.</li>
		<li>Once infusion of the PCL solution has begun, turn on the voltage source and set to a 20 kV operating voltage.</li>
		<li>Infuse the solution for 12 min to create a homogenous cuff from 0.5 ml of the PCL solution. 
		NOTE: On average, each cuff has enough electrospun material to create two six-strip sheets, which can be used to create a total of three four-layered scaffolds.</li>
	</ol>
	</li>
	<li>Laser cut the PCL cuff to form multiple small sheets on a VersaLaser Cutter 2.3 operated at low-vacuum setting, 10.0 keV landing voltage, 6.4 mm working distance, and probe diameter of 3.0. 
	NOTE: In this example, a computer aided design was used to instruct the cutter to yield multiple sheets of 1.5 mm x 35 mm x 150 &#181;m scaffolds with evenly distributed 150 &#181;m diameter holes at 15% pore area.</li>
	<li>Plasma etch the PCL scaffolds using a plasma cleaner to induce hydrophilicity of the PCL surface with ion acceleration. Set the vacuum to 450 mTorr and the treat the scaffolds for 30 sec at high power (29.6 W).</li>
	<li>Bathe the scaffolds in 70% ethanol in a sterile environment.</li>
	<li>Coat the individual scaffolds with collagen to facilitate cellular adhesion and proliferation in vivo.
	<ol>
		<li>Create a collagen coating solution by diluting a 8:1:2.5 sterile solution of Purecol collagen 3 mg/ml standard solution, 10x PBS and 0.1 N NaOH 1:9 in 1x PBS at 4 &#176;C. Mix thoroughly to ensure solution homogeneity.</li>
		<li>Coat individual 1.5 mm x 35 mm x 150 &#181;m scaffolds with a thin layer film of the above collagen solution. Allow to dry for 24 hr in a sterile environment.</li>
	</ol>
	</li>
	<li>Using 5-0 Vicryl sutures, stack and affix four individual 1.5 mm x 35 mm x 150 &#181;m scaffolds using a Krackow stitch to create a final 0.6 mm thick, multi-layered, collagen-coated scaffold that is ready for implantation.</li>
</ol>
2. Rat Surgery Protocol
<ol>
	<li>Induce anesthesia by placing rat in inhalation chamber and delivering 2% isoflurane with 2 L/min oxygen. Confirm the rat is adequately anesthetized by applying pressure to hind foot and evaluating for any response.</li>
	<li>On the non-sterile back table, subcutaneously inject 25 mg/kg ampicillin and 0.03 mg/kg buprenorphine.</li>
	<li>Apply ophthalmic ointment to the eyes. Clip the fur from the operative hind limb and prep the surgical site with three alternating scrubs of chlorhexidine and 70% ethanol.</li>
	<li>Transfer the rat to the operating table, on a heated pad to prevent hypothermia. Secure nose cone, and maintain anesthesia through the procedure with 2% isoflurane in 2 L/min oxygen, delivered nose cone. Drape in a sterile manner, leaving the operative limb exposed.&#160;</li>
	<li>Before starting the surgery, first confirm adequate anesthetization by lack of a response to hind foot pressure.</li>
	<li>Make a 2 cm long vertical incision medial to the knee, centered at the level of the patella. Retract the skin laterally until the incision is centered over the knee.</li>
	<li>Use a scalpel to make a medial parapatellar arthrotomy by cutting just medial to the patella and extending proximally to the level of the musculotendinous junction of the quadriceps and distally to the level of the patellar tendon insertion on the tibial tubercule. Take care not to cut the patellar or quadriceps tendons.</li>
	<li>Release patella laterally by making a 1 cm vertical incision through the knee capsule just lateral to the patellar tendon.</li>
	<li>Ensure the knee is extended. Take a pair of fine scissors and pass under the patella from lateral to medial. Spread the scissors a couple times so that the extensor mechanism can be translated to either side.</li>
	<li>While flexing the knee, translate the patella laterally to expose the inside of the knee joint. Ensure clear visualization of the intercondylar notch and femoral condyles. Using a scalpel, transect the ACL and PCL in the notch.</li>
	<li>Load power drill with a 1.6 mm k-wire. Place k-wire tip on ACL origin in the intercondylar notch. Drill superolaterally and visualize the exit point on the lateral aspect of the femur, removing any soft tissue as necessary with a scalpel. Pass the k-wire in and out a few times to ensure clear passage for the graft.</li>
	<li>Place the k-wire on the ACL footprint on the tibial plateau. Drill anterolaterally and visualize exit point on the anterolateral proximal tibia. Use scalpel to clear soft tissue as necessary so that the point where the k-wire exits the tibia is fully visualized.</li>
	<li>Pass a shortened Keith needle (ideally no more than 2 inches long) through the femoral bone tunnel. Thread the two suture ends from one end of the graft through the eye of the Keith needle. Use the needle to pull one end of the graft through the femoral tunnel.</li>
	<li>Repeat the prior step to pass the other end of the graft through the tibial tunnel.</li>
	<li>Use 4-0 Vicryl suture to affix the femoral end of the graft to surrounding periosteum or other soft tissue with a figure-of-eight stitch. Manually tension the graft with the knee in extension. Affix the tibial end of the graft to surrounding periosteum or other soft tissue with a figure-of-eight stitch.</li>
	<li>Use a scissor to cut off excess graft on both ends, leaving 1-2 mm on each end past the figure-of-eight stitch.</li>
	<li>Extend the knee and reduce the patella. Using 4-0 vicryl, place a single figure-of-eight stitch to close the medial joint capsule, preventing lateral subluxation of the patella.</li>
	<li>Close the skin with a running subcuticular 5-0 Monocryl or Vicryl suture, with care not to suture the underlying muscle or have any visible suture once the skin is closed.</li>
	<li>Inject rats subcutaneously with buprenorphine every 12 hr for a total of three days postoperatively. Check surgical site for any drainage or wound dehiscence at the time of injection. Limping and some swelling are normal in the first few days after surgery, but promptly address any postoperative concerns in conjunction with veterinary staff. &#160;The animal may be return to social housing at 2 weeks postoperatively, when surgical incisions are completely healed.</li>
</ol>
3. Data Collection Protocol
<ol>
	<li>At the time of sacrifice, asphyxiate rats individually in a closed CO2 chamber followed by thoracotomy.</li>
	<li>Harvest both the surgically reconstructed and contralateral limbs by separating at the hip joint.
	<ol>
		<li>For the reconstructed limbs, remove all soft tissue, including the posterior cruciate ligament and the remnants from the surgically disrupted native ACL, by fine dissection to isolate only the femur, tibia, and graft.</li>
		<li>For the contralateral limbs, remove all soft tissue except the native ACL as well as the femur and tibia by fine dissection.</li>
		<li>Use a rotary tool such as Dremel, to remove all but &#190; to 1 cm of bone from each end of the femur-graft-tibia complex.</li>
		<li>During this process and throughout biomechanical testing, regularly and frequently spray the ligamentous regions with normal saline to prevent desiccation of the harvested knee which may falsely alter results.</li>
	</ol>
	</li>
	<li>For histological analysis, fix each knee individually in 4% paraformaldehyde solution at 25 &#176;C for 48 hr. Next, submerge the knee in a solution of Immunocal reagent for complete decalcification; this process is dependent on the calcific contents of the sample and may take up to five days. Check individual samples daily to assess progress as incomplete decalcification can lower sample quality. Once complete, perform sectioning, slide mounting, and staining as desired.</li>
	<li>Perform biomechanical testing to assess the functional capacity of the tissue engineered ligament.
	<ol>
		<li>Secure the femur and tibia by wrapping 28 G&#160;galvanized steel wire around the epiphysis of each bone separately. This is to prevent inaccurate biomechanical testing data from premature tensile failure of the sample at the bone rather than at the ligament of interest.</li>
		<li>Pot the femur into a mixture of polymethylmethacrylate (PMMA) bone cement. To do this, mix the two cement components and immediately use the viscous mixture to secure the femur into the metal, completely encasing the diaphysis of the bone in the cemented pot with the epiphysis and attached ligament protruding freely. Allow spontaneous free radical polymerization to gradually transform the mixed viscous components to a doughy material and eventually into a solid hardened matrix. 
		NOTE: This process takes several minutes and can be monitored by manually assessing the temperature of a bolus made from the remaining cement; the temperature should transiently increase during the exothermic polymerization reaction and subside to RT after the material solidifies.</li>
		<li>Repeat the same process above for cementing the tibia, except while maintaining the knee ligament at 20&#176; flexion for ideal mechanical testing.</li>
		<li>Mount the cemented femur-graft-tibia complex onto a tensile testing apparatus, and prepare to record load and displacement as a function of time from the beginning of tension to failure. In this example, we used an Instron Model 5564 with a 1 kN load cell.</li>
		<li>Pre-tension the graft to 2 N at a ramp rate of 0.5 N/min and then test the graft to failure at a strain rate of 0.5 mm/sec. During the process, be sure that the ligament is failing at the mid-substance and that the bony femur and tibia are secure and not prematurely failing, which may inaccurately assess biomechanical properties of the tested ligament.</li>
		<li>Use the generated load-displacement curves to compute the failure load and stiffness of the tested ligament.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Fetto, J. F., Marshall, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+and+diagnosis+of+anterior+cruciate+ligament+insufficiency.">The natural history and diagnosis of anterior cruciate ligament insufficiency.</a> Clin Orthop Relat Res. (147), 29-38 (1980).</li><li>Kim, Y. M., Lee, C. A., Matava, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+results+of+arthroscopic+single-bundle+transtibial+posterior+cruciate+ligament+reconstruction:+a+systematic+review.">Clinical results of arthroscopic single-bundle transtibial posterior cruciate ligament reconstruction: a systematic review.</a> Am J Sports Med. 39 (2), 425-434 (2011).</li><li>Andersson, C., Odensten, M., Gillquist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knee+function+after+surgical+or+nonsurgical+treatment+of+acute+rupture+of+the+anterior+cruciate+ligament:+a+randomized+study+with+a+long-term+follow-up+period.">Knee function after surgical or nonsurgical treatment of acute rupture of the anterior cruciate ligament: a randomized study with a long-term follow-up period.</a> Clin Orthop Relat Res. (264), 255-263 (1991).</li><li>Klimkiewicz, J. J., Petrie, R. S., Harner, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+treatment+of+combined+injury+to+anterior+cruciate+ligament,+posterior+cruciate+ligament,+and+medial+structures.">Surgical treatment of combined injury to anterior cruciate ligament, posterior cruciate ligament, and medial structures.</a> Clin Sports Med. 19 (3), 479-492 (2000).</li><li>Petrigliano, F. A., McAllister, D. R., Wu, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+for+anterior+cruciate+ligament+reconstruction:+a+review+of+current+strategies.">Tissue engineering for anterior cruciate ligament reconstruction: a review of current strategies.</a> Arthroscopy. 22 (4), 441-451 (2006).</li><li>Groot, J. 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=Use+of+porous+polyurethanes+for+meniscal+reconstruction+and+meniscal+prostheses.">Use of porous polyurethanes for meniscal reconstruction and meniscal prostheses.</a> Biomaterials. 17 (2), 163-173 (1996).</li><li>Leong, N. L., Petrigliano, F. A., McAllister, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+tissue+engineering+strategies+in+anterior+cruciate+ligament+reconstruction.">Current tissue engineering strategies in anterior cruciate ligament reconstruction.</a> J Biomed Mater Res A. 102 (5), 1614-1624 (2014).</li><li>Duling, R. R., Dupaix, R. B., Katsube, N., Lannutti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+electrospun+polycaprolactone+(PCL):+a+potential+scaffold+for+tissue+engineering.">Mechanical characterization of electrospun polycaprolactone (PCL): a potential scaffold for tissue engineering.</a> J Biomech Eng. 130 (1), 011006 (2008).</li><li>Shao, Z., 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=Polycaprolactone+electrospun+mesh+conjugated+with+an+MSC+affinity+peptide+for+MSC+homing+in+vivo.">Polycaprolactone electrospun mesh conjugated with an MSC affinity peptide for MSC homing in vivo.</a> Biomaterials. 33 (12), 3375-3387 (2012).</li><li>Tillman, B. 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=The+in+vivo+stability+of+electrospun+polycaprolactone-collagen+scaffolds+in+vascular+reconstruction.">The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction.</a> Biomaterials. 30 (4), 583-588 (2009).</li><li>Wise, S. G., 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+multilayered+synthetic+human+elastin/polycaprolactone+hybrid+vascular+graft+with+tailored+mechanical+properties.">A multilayered synthetic human elastin/polycaprolactone hybrid vascular graft with tailored mechanical properties.</a> Acta Biomater. 7 (1), 295-303 (2011).</li><li>Vargel, I., Korkusuz, P., Mencelo&#287;lu, Y. Z., Pi&#351;kin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+performance+of+antibiotic+embedded+electrospun+PCL+membranes+for+prevention+of+abdominal+adhesions.">In vivo performance of antibiotic embedded electrospun PCL membranes for prevention of abdominal adhesions.</a> J Biomed Mater Res B Appl Biomater. 81 (2), 530-543 (2007).</li><li>Cao, H., McHugh, K., Chew, S. Y., Anderson, 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=The+topographical+effect+of+electrospun+nanofibrous+scaffolds+on+the+in+vivo+and+in+vitro+foreign+body+reaction.">The topographical effect of electrospun nanofibrous scaffolds on the in vivo and in vitro foreign body reaction.</a> J Biomed Mater Res A. 93 (3), 1151-1159 (2010).</li><li>Joshi, V. S., Lei, N. Y., Walthers, C. M., Wu, B., Dunn, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroporosity+enhances+vascularization+of+electrospun+scaffolds.">Macroporosity enhances vascularization of electrospun scaffolds.</a> J Surg Res. 183 (1), 18-26 (2013).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+poly(epsilon-caprolactone)+microfiber+and+multilayer+nanofiber/microfiber+scaffolds:+characterization+of+scaffolds+and+measurement+of+cellular+infiltration.">Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.</a> Biomacromolecules. 7 (10), 2796-2805 (2006).</li><li>Vaz, C. M., van Tuijl, S., Bouten, C. V., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+scaffolds+for+blood+vessel+tissue+engineering+using+a+multi-layering+electrospinning+technique.">Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique.</a> Acta Biomater. 1 (5), 575-582 (2005).</li><li>Kawamura, S., Ying, L., Kim, H. J., Dynybil, C., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+accumulate+in+the+early+phase+of+tendon-bone+healing.">Macrophages accumulate in the early phase of tendon-bone healing.</a> J Orthop Res. 23 (6), 1425-1432 (2005).</li><li>Hays, P. L., 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+role+of+macrophages+in+early+healing+of+a+tendon+graft+in+a+bone+tunnel.">The role of macrophages in early healing of a tendon graft in a bone tunnel.</a> J Bone Joint Surg Am. 90 (3), 565-579 (2008).</li><li>Dagher, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+modulates+macrophage+accumulation+in+tendon-bone+healing.">Immobilization modulates macrophage accumulation in tendon-bone healing.</a> Clin Orthop Relat Res. 467 (1), 281-287 (2009).</li><li>Bedi, 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=Effect+of+early+and+delayed+mechanical+loading+on+tendon-to-bone+healing+after+anterior+cruciate+ligament+reconstruction.">Effect of early and delayed mechanical loading on tendon-to-bone healing after anterior cruciate ligament reconstruction.</a> J Bone Joint Surg Am. 92 (14), 2387-2401 (2010).</li><li>Bedi, A., Kawamura, S., Ying, L., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+tendon+graft+healing+between+the+intra-articular+and+extra-articular+ends+of+a+bone+tunnel.">Differences in tendon graft healing between the intra-articular and extra-articular ends of a bone tunnel.</a> HSS J. 5 (1), 51-57 (2009).</li><li>Fu, S. C., 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=Effect+of+graft+tensioning+on+mechanical+restoration+in+a+rat+model+of+anterior+cruciate+ligament+reconstruction+using+free+tendon+graft.">Effect of graft tensioning on mechanical restoration in a rat model of anterior cruciate ligament reconstruction using free tendon graft.</a> Knee Surg Sports Traumatol Arthrosc. 21 (5), 1226-1233 (2013).</li><li>Fan, H., Liu, H., Wong, E. J., Toh, S. L., Goh, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+study+of+anterior+cruciate+ligament+regeneration+using+mesenchymal+stem+cells+and+silk+scaffold.">In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold.</a> Biomaterials. 29 (23), 3324-3337 (2008).</li><li>Landis, J. R., Koch, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+observer+agreement+for+categorical+data.">The measurement of observer agreement for categorical data.</a> Biometrics. 33 (1), 159-174 (1977).</li><li>Joshi, S. M., Mastrangelo, A. N., Magarian, E. M., Fleming, B. C., Murray, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-platelet+composite+enhances+biomechanical+and+histologic+healing+of+the+porcine+anterior+cruciate+ligament.">Collagen-platelet composite enhances biomechanical and histologic healing of the porcine anterior cruciate ligament.</a> Am J Sports Med. 37 (12), 2401-2410 (2009).</li><li>Leong, N. L., 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=In+vitro+and+in+vivo+evaluation+of+heparin+mediated+growth+factor+release+from+tissue-engineered+constructs+for+anterior+cruciate+ligament+reconstruction.">In vitro and in vivo evaluation of heparin mediated growth factor release from tissue-engineered constructs for anterior cruciate ligament reconstruction.</a> J Orthop Res. 10, (2014).</li><li>Seo, Y. K., 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=Increase+in+cell+migration+and+angiogenesis+in+a+composite+silk+scaffold+for+tissue-engineered+ligaments.">Increase in cell migration and angiogenesis in a composite silk scaffold for tissue-engineered ligaments.</a> J Orthop Res. 27 (4), 495-503 (2009).</li><li>Freeman, J. W., Woods, M. D., Laurencin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+the+anterior+cruciate+ligament+using+a+braid-twist+scaffold+design.">Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design.</a> J Biomech. 40 (9), 2029-2036 (2007).</li><li>Bashur, C. A., Shaffer, R. D., Dahlgren, L. A., Guelcher, S. A., Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fiber+diameter+and+alignment+of+electrospun+polyurethane+meshes+on+mesenchymal+progenitor+cells.">Effect of fiber diameter and alignment of electrospun polyurethane meshes on mesenchymal progenitor cells.</a> Tissue Eng Part A. 15 (9), 2435-2445 (2009).</li><li>Dash, T. K., Konkimalla, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly-&#1108;-caprolactone+based+formulations+for+drug+delivery+and+tissue+engineering:+A+review.">Poly-&#1108;-caprolactone based formulations for drug delivery and tissue engineering: A review.</a> J Control Release. 158 (1), 15-33 (2012).</li><li>Yoshimoto, H., Shin, Y. M., Terai, H., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biodegradable+nanofiber+scaffold+by+electrospinning+and+its+potential+for+bone+tissue+engineering.">A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering.</a> Biomaterials. 24 (12), 2077-2082 (2003).</li><li>Xu, Y., Ao, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+biomechanical+studies+of+inter-strand+healing+in+four-strand+autograft+anterior+cruciate+ligament+reconstruction+in+a+rabbit+model.">Histological and biomechanical studies of inter-strand healing in four-strand autograft anterior cruciate ligament reconstruction in a rabbit model.</a> Knee Surg Sports Traumatol Arthrosc. 17 (7), 770-777 (2009).</li><li>Shino, K., 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=Replacement+of+the+anterior+cruciate+ligament+by+an+allogeneic+tendon+graft.+An+experimental+study+in+the+dog.">Replacement of the anterior cruciate ligament by an allogeneic tendon graft. An experimental study in the dog.</a> J Bone Joint Surg Br. 66 (5), 672-681 (1984).</li><li>Stasiak, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+device+to+apply+controlled+flexion+and+extension+to+the+rat+knee+following+anterior+cruciate+ligament+reconstruction.">A novel device to apply controlled flexion and extension to the rat knee following anterior cruciate ligament reconstruction.</a> J Biomech Eng. 134 (4), 041008 (2012).</li><li>Brophy, R. 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=Effect+of+short-duration+low-magnitude+cyclic+loading+versus+immobilization+on+tendon-bone+healing+after+ACL+reconstruction+in+a+rat+model.">Effect of short-duration low-magnitude cyclic loading versus immobilization on tendon-bone healing after ACL reconstruction in a rat model.</a> J Bone Joint Surg Am. 93 (4), 381-393 (2011).</li></ol>

The authors declare that they have no competing financial interests.

ACL injuries are a common condition in orthopaedic sports surgery, with limited options for reconstruction at the present time. In order to develop an appropriate tissue-engineered substitute for the ACL that will allow regeneration in vivo, a suitable animal model is required. In this study, the fabrication of a biodegradable engineered graft is described, as is its in vivo implantation using a reproducible model of ACL reconstruction in an athymic rat. This model can be utilized to evaluate different tissue-engineered ACL grafts composed of various biomaterials, including cellular grafts and those with incorporated growth factors.
In this particular study, we tested an acellular, electrospun polycaprolactone graft with aligned fibers. Previous studies of tissue engineered ligament reconstruction have implanted braided or extruded grafts made of a variety of materials including silk, PLLA, and polyurethane27-29. None of these materials resulted in both successful integration of the graft and recapitulation of the mechanical properties of ACL7. While many polymers have the potential for use in ligament reconstruction, this study investigated PCL because it is biologically inert, non-toxic, degrades slowly in vivo, and is easily manufactured into a desired conformation30. PCL is also mechanically robust and shows little plastic deformation under mechanical stress30. Its use has been established in the bone tissue engineering literature as a reliable reservoir for mineralization and type I collagen deposition due to its aligned nanofiber structure when electrospun31. Also, it has been shown that the high surface to volume and short diffusion length scale of the small diameter fibers in electrospun PCL mats are favorable for controlled drug delivery and use in tissue engineering31.
It was found that the implanted grafts were biocompatible based on a lack of clinical adverse reaction, and facilitated native cell infiltration as seen histologically at 16 weeks post-operatively. We utilized an athymic animal model in this study as it is the first stage of a two-stage project that will eventually utilize human cells implanted with the graft, and an athymic model would reduce concerns regarding rejection of the human cells. The electrospun polymer scaffold facilitated both cell and matrix alignment in the regenerated ACL. As seen in the cross-section of the graft, the majority of the cells were aligned in the direction of the fibers. The implanted grafts showed increased mechanical properties over time. Load to failure of the polymer grafts doubled as compared to the reconstructed ACL immediately postoperatively. While the peak load and stiffness of the PCL graft may seem low in comparison to native ACL, it is important to remember that these results should be viewed in light of the fact that even the current gold standard, autografts or allografts, are not able to achieve the mechanical strength of healthy ACL by 16 weeks postoperatively. For example, Xu et al. reported on an ACL autograft in a rabbit model, in which peak load was 20%-35% and stiffness was 23%-36% that of healthy native ACL by 6 months postoperatively32. Additionally, an allograft study in a canine model demonstrated approximately 30% the peak load and 40% the stiffness of native ACL by 30 weeks postoperatively33.
While it is outside the scope of this paper, many other analyses may be performed to evaluate graft quality after use of this animal model. This includes but is not limited to bioluminescent imaging or X-rays and CT scans in vivo, and a multitude of stains and assays such as immunohistochemistry for collagen markers or inflammatory markers. For instance, we previously published results on the quantification of aligned collagen fibers using picrosirius red staining after implantation of electrospun PCL scaffolds implanted in this animal model26.
Potential limitations of this study include the choice of the animal model itself. The inherent differences in anatomy and gait in the quadrupedal rat compared to bipedal humans mean that the biomechanics of the ACL do vary and that translation of clinical parameters between models should be done with knowledge of these limitations. However, this issue is common in animal studies and does not negate the importance or translational potential of this research.
There has been interesting research on post-operative protocols for rat ACL reconstruction using autograft that may be applied to our model for engineered ACL replacements in the future. External fixation devices have been used to immobilize rats post-operatively in order to allow improved tendon-bone healing in a tendon autograft model19. Additionally, it has been shown that delayed cyclic loading, such as with the flexion-extension device described by Stasiak et al.34, may further enhance autograft incorporation20. However, it has also been shown that short duration low magnitude cyclic loading can also cause increased inflammation and decreased bone formation at the bone-tendon interface35. Further investigation must be conducted to evaluate the applicability of these findings to an electrospun, polymer-based ACL replacement, as such a graft would have weaker initial mechanical properties than a tendon allograft.
The present study has developed a model of ACL reconstruction with an acellular electrospun graft in an athymic rat, based off modifications of a previously described rat autograft model17-22. We demonstrated the elaboration of dense-aligned collagen throughout the graft with a concurrent improvement of load to failure of the graft over time. This study also provides proof of concept for employing this model in the future to evaluate various tissue-engineered grafts for ACL reconstruction. In particular, the athymic rat allows for the seeding of xenogeneic donor cells.

Rupture of the anterior cruciate ligament (ACL) is one of most common ligament injuries of the knee1. Because the ruptured ACL&#8217;s is unable to heal without surgical intervention, the limitations in activities of daily living as well as participation in sports drive over 175,000 patients to undergo surgery each year2, with an estimated cost of one billion dollars annually3. Currently, either autograft or allograft tendon is used for ligament reconstruction. Though high success rates can be achieved with both autograft and allograft replacement, serious complications are associated with these reconstruction options4. Autograft tissue is associated with donor site morbidity and is limited in supply, especially in instances of re-rupture or multi-ligamentous injury. On the other hand, allograft tissue is linked with delayed graft integration, adverse inflammatory response, theoretical infectious risk, and limited supply5. Synthetic non-degradable grafts were developed in the 1970s and 1980s but were hampered by premature graft rupture, foreign body reactions, osteolysis, and synovitis6. As a result of these serious concerns, there are currently no synthetic grafts available for clinical use in the United States.
Due to these limitations with existing graft options and to recent developments in biology, engineering, and regenerative medicine, there has been great interest in a tissue engineered solution for ACL grafting. Current tissue engineering strategies employ degradable biological and synthetic materials to allow for host tissue ingrowth while avoiding the limitations associated with permanent synthetic material implantation7.
Polycaprolactone (PCL) is a biodegradable polymer that is FDA approved for a number of medical applications including adhesion barrier and wound dressing8, that has been used in a wide variety of applications including vascular, bone, cartilage, nerve, skin, and esophageal tissue engineering5,9-16. Favorable biocompatibility, relatively long in-vivo half-life, adequate mechanical strength, and high elasticity contribute to the popularity of this polymer in tissue engineering. In a rodent model of wound healing, implanted electrospun PCL was shown to be non-immunogenic and to integrate into local tissue without adverse reactions13. An SEM image of electrospun PCL is shown in Figure 1.
With current FDA regulatory standards, efficacy and safety in both small and large animal models would be required for a PCL or any other engineered ACL graft to move into clinical trials in the United States. Additionally, in vivo conditions can often augment the properties of an in vitro tissue engineered ACL graft. A rat model of autologous ACL reconstruction with flexor digitorum longus tendon has been previously described, in which the native ACL was severed, femoral and tibial tunnels were drilled, and the graft was passed and secured in place with suture17-22. In this paper, we will describe a modification of this model for the evaluation of engineered ACL replacements rather than for autograft-based reconstruction (Figure 2).
Although many animal models exist for ligament tissue engineering, the rat is advantageous compared to larger models for a number of reasons. These advantages include easier husbandry and handling, fewer ethical considerations, and reduced cost17,23. In addition, the rat model has been used extensively as a model for orthopaedic tissue regeneration, including cartilage, tendon, and bone tissue engineering24. In particular, athymic nude rats were chosen due to their lack of cell-mediated immune response25, allowing for the eventual implantation of xenogeneic donor cells in this model to further enhance the engineered graft in the future. In this methods paper, we describe the fabrication and surgical implantation of an acellular, biodegradable polymer graft in an athymic rat model of ACL reconstruction.

<table><tbody><tr><td>Medical grade ester terminated poly (&amp;epsilon;-caprolactone), granule form (MW = 110,000)</td><td><a href="http://www.absorbables.com" target="_blank"><img src="/img/new_window.png" /> Lactel Absorbable Polymers</a></td><td></td><td>Custom synthesized polymer to desired molecular weight</td></tr><tr><td>1,1,1,3,3,3-hexafluoro-2-propanol</td><td>Sigma-Aldrich</td><td>105228</td><td>Solvent for PCL polymer</td></tr><tr><td>18 G x 1&amp;#189;&quot; bevel needle</td><td>BD Medical</td><td>305196</td><td></td></tr><tr><td>Remote Infuse/Withdraw Programmable Syringe Pump</td><td>Harvard Apparatus</td><td>702101</td><td></td></tr><tr><td>VersaLaser VLS2.30 Laser Engraver</td><td>Microgeo USA</td><td>VLS2.30</td><td></td></tr><tr><td>Expanded Plasma Cleaner 115&amp;nbsp;V</td><td>Harrick Plasma</td><td>PDC-001</td><td>Plasma etch just prior to collagen coating for surface modification</td></tr><tr><td>PureCol Collagen Standard Solution, 3&amp;nbsp;mg/ml</td><td>Advanced Biomatrix</td><td>5015-A</td><td>Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1&amp;nbsp;N NaOH 1:9 in 1x PBS</td></tr><tr><td>Suture, 5-0 Vicryl</td><td>Henry Schein</td><td>1086471</td><td></td></tr><tr><td>Suture, 4-0 Vicryl</td><td>Henry Schein</td><td>6540072</td><td></td></tr><tr><td>Sharp-pointed Dissecting Scissors (Straight; 4.5&amp;nbsp;inch)</td><td>Fisher Scientific</td><td>8940</td><td></td></tr><tr><td>Buphrenorphine hydrochloride</td><td>Sigma-Aldrich</td><td>B9275</td><td>Use 0.03 mg/kg for both intra- and post-operatively for pain control</td></tr><tr><td>Ampicillin, injectable</td><td>Henry Schein</td><td>1185678</td><td>Use 25 mg/kg subcutaneously during the procedure</td></tr><tr><td>K-wire, 1.6 mm</td><td>Spectrum Surgical</td><td>SI040062</td><td></td></tr><tr><td>Keith Needle, Straight 1&amp;#189;&quot;</td><td>Delasco Dermatology Lab &amp;amp; Supply</td><td>KE-112</td><td></td></tr><tr><td>Immunocal Decalcifying Solution</td><td>Fisher Scientific</td><td>NC9491030</td><td></td></tr><tr><td>Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td><td>US Dental Depot</td><td>OPTICRYL 100410&amp;nbsp;</td><td></td></tr><tr><td>Instron Model 5564 Tensile Testing Machine</td><td>Instron</td><td>5564</td><td>Any comparable tensile testing apparatus is suitable</td></tr></tbody></table>

athymic rat model for evaluation of engineered anterior cruciate ligament grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

In our experience of 92 rat surgeries by a single surgeon, mean operative time from incision to completion of wound was 16.9 min, with a standard deviation of 4.7 min. At the time of sacrifice, rats weighed 356 &#177; 23 g. All rats tolerated the surgery well, and experienced no complications. Immediately after surgery, the rats were noted to bear weight on the operative extremity, but exhibited a slight limp. By one week post-operatively, all rats were ambulating with no appreciable limp. The animals all gained weight steadily during the course of the study, with no observed abnormalities in feeding, urination, or defecation habits. Clinically, no gross wound dehiscence, erythema, swelling, effusion, or drainage was observed postoperatively.
The 92 rat surgeries mentioned above were not performed primarily for the purpose of this methods manuscript. Rather, they were used to test various engineered graft conditions. While the detailed mechanical testing and histological results are outside the scope of this paper, more details can be found in a paper by Leong et al.26. In brief, at 16 weeks following the reconstruction, histological analysis of the sectioned knee demonstrated that the scaffold matrix became largely infiltrated by fibroblasts secreting eosinophilic collagen with good integration into the bone tunnels (Figure 3). At this time, the scaffold has been completely resorbed and no evidence of the polymer was visualized. Additionally, immunohistochemistry for the macrophage marker CD68 demonstrated minimal inflammatory response at 16 weeks post-operatively (Figure 4).
Biomechanical properties were assessed immediately after sacrifice. All tested samples failed at the mid-substance (Figure 5). Using load-displacement curves generated from tensile testing (Figure 6), failure load and stiffness were computed for each group. At 16 weeks post-implantation, the electrospun polymer graft had approximately double the peak load and stiffness of the graft tested immediately post-implantation, but these values were lower than the native ACL26.
<img alt="Figure 1" src="/files/ftp_upload/52797/52797fig1.jpg" /> 
Figure 1. SEM image of electrospun polycaprolactone scaffold with aligned fibers. <a href="https://www.jove.com/files/ftp_upload/52797/52797fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52797/52797fig2.jpg" /> 
Figure 2. Athymic rat model of ACL reconstruction.&#160;(A) Isolation of the patellar tendon via a medial parapatellar skin incision. (B) Drilling of femoral tunnel using 1.6 mm k-wire. (C) Drilling of tibial tunnel. (D) Placement of 1.2 mm Keith needle through femoral tunnel to pull graft through. (E) Electrospun polycaprolactone graft pulled through femoral tunnel. (F) Graft pulled through both femoral and tibial tunnels, before the ends are trimmed and sutured to periosteum, and a layered closure is performed. <a href="https://www.jove.com/files/ftp_upload/52797/52797fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52797/52797fig3.jpg" /> 
Figure 3. Hematoxylin and eosin staining of electrospun polymer graft (top) at tibial bone tunnel (left), midsubstance (center), and femoral bone tunnel (right). For comparison, native ACL is shown(bottom), at tibial insertion (left), midsubstance (center) and femoral origin (right), 10X. <a href="https://www.jove.com/files/ftp_upload/52797/52797fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52797/52797fig4.jpg" /> 
Figure 4. Colorimetric immunohistochemistry staining for CD68, a marker for macrophages. Qualitatively, there appears to be slightly more positive staining in the bone tunnel at 8 weeks than in the intra-articular region of the graft or at 16 weeks post-op. There appears to be minimal inflammation in the grafts. All images are 20X magnification. <a href="https://www.jove.com/files/ftp_upload/52797/52797fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52797/52797fig5.jpg" /> 
Figure 5. Images of mechanical testing of implanted electrospun graft, demonstrating failure at midsubstance of graft. <a href="https://www.jove.com/files/ftp_upload/52797/52797fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52797/52797fig6.jpg" /> 
Figure 6. Sample load-displacement curve for tissue-engineered ACL graft at 16 weeks after implantation in athymic rat model. <a href="https://www.jove.com/files/ftp_upload/52797/52797fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts at JoVE.com

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.3K Views.  University of California Los Angeles. Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Video: Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

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.

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. ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Medicine

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

Biology

Impact of Non-Invasive ACL Injury and Surgery on Protease Activity in Mice

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

Author Spotlight: Investigating Early Events and Long-Term Effects of ACL Injuries for Osteoarthritis Progression

Non-invasive ACL injury is a reliable and clinically relevant method for initiating post-traumatic osteoarthritis (PTOA) in mice. This injury method also allows for in vivo quantification of protease activity in the joint at early time points post-injury using protease-activatable near infrared probes and fluorescence reflectance imaging.

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within ...

A Mouse Model of Ankle-Subtalar Complex Joint Instability

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and can eventually lead to post-traumatic osteoarthritis (PTOA) in the long term. However, due to the complexity of the injury mechanism and the clinical manifestations, such as ecchymosis, hematoma, or tenderness in the lateral foot, there is no clinical consensus on diagnosing and treating ASCJ instability. Since the musculoskeletal structure of the bones and ligaments of the mouse hindfoot is comparable to that of humans, an animal model of ASCJ instability in mice was established by the transection of ligaments around the ASCJ. The model was well-validated through a series of behavioral tests and histological analyses, including a balance beam test, a footprint analysis (an assessment of exercise level and balance ability in mice), a thermal nociception assessment (an assessment of foot sensory function in mice), micro-computed tomography (CT) scanning, and section staining of the articular cartilage (an assessment of articular cartilage damage and degeneration in mice). The successful establishment of a mouse model of ASCJ instability will provide a valuable reference for clinical research on the injury mechanism and result in better treatment options for ankle sprain.

The ankle-subtalar complex joint (ASCJ) is the core of the foot and plays a key role in balance control in daily activities. Sports injuries often lead to instability in this joint. Here, we describe a mouse model of ligament transection-induced instability of the ASCJ.

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and ...

Visualization of Knee Joint Degeneration after Non-invasive ACL Injury in Rats

Source: Lindsey K. Lepley1,2, Steven M. Davi1, Timothy A. Butterfield3,4 and Sina Shahbazmohamadi5,
1Department of Kinesiology, University of Connecticut, Storrs, CT; 2Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT; 3Department of Rehabilitation Sciences, University of Kentucky, Lexington, KY; 4Center for Muscle Biology, Department of Physiology, University of Kentucky, Lexington, KY; 5Biomedical Engineering Department, University of Connecticut, Storrs, CT
Anterior cruciate ligament (ACL) injury to the knee dramatically increases the risk of post-traumatic osteoarthritis (PTOA), as approximately one-third of individuals will demonstrate radiographic PTOA within the first decade following ACL injury. Though ACL reconstruction (ACLR) successfully restores knee joint stability, ACLR and current rehabilitation techniques do not prevent the onset of PTOA. Therefore, ACL injury represents the ideal model to study the development of PTOA after traumatic joint injury.
Rat models have been used extensively to study the onset and effect of ACL injury on PTOA. The most widely used model of ACL injury is ACL transection, which is an acute model that surgically destabilizes the joint. Though practical, this model does not faithfully mimic human ACL injury due to the invasive and non-physiological injury procedures that mask the native biological response to injury. To improve the clinical translation of our results, we have recently developed a novel non-invasive model of ACL injury where the ACL is ruptured through a single load of tibial compression. This injury closely replicates injury conditions relevant to humans and is highly reproducible.
Visualization of joint degeneration through micro-computed tomography (&#181;CT) provides several major advancements over traditional OA staining techniques, including rapid, high-resolution, non-destructive 3D imaging of whole joint degeneration. The goal of this demonstration is to introduce the state of the art non-invasive ACL injury in a rodent model and use &#181;CT to quantify knee joint degeneration.

Knee Joint Degeneration after Non-invasive Anterior Cruciate Ligament Injury

Source: Lindsey K. Lepley1,2, Steven M. Davi1, Timothy A. Butterfield3,4 and Sina Shahbazmohamadi5,
1Department of Kinesiology, University of Connecticut, ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Summary

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Chapters in this video

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

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

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

A Mouse Model of Ankle-Subtalar Complex Joint Instability

Visualization of Knee Joint Degeneration after Non-invasive ACL Injury in Rats