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

Los autores desean agradecer a Gabriel Arom y Michael Yeranosian por sus contribuciones técnicas a las iteraciones anteriores de este proyecto. Este proyecto fue financiado por el OREF Clínico Científico Training Grant (NL), HH Lee quirúrgica Beca de Investigación (NL), la Administración de Veteranos BLR + D Revisión Mérito 1 I01 BX00012601 (DM) y Musculoesqueléticas Trasplante Fundación Premio al Investigador Joven (FP).

NOTA: Todas las cirugías animales fueron aprobadas por el personal y animal veterinaria comité uso local antes de comenzar los experimentos. 1. Preparación de electrohiladas policaprolactona Andamios <ol><li> Pesar y disolver éster de grado médico terminado poli (ε-caprolactona) en forma de gránulos en 1,1,1,3,3,3-hexafluoro-2-propanol para crear un 10% w / w solución del polímero PCL. Deje que el revuelo solución utilizando una placa de agitación durante al menos 3 horas para a...

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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. 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Las lesiones del LCA son una condición común en la cirugía ortopédica deportes, con opciones limitadas para la reconstrucción en la actualidad. A fin de desarrollar un sustituto de tejido de ingeniería apropiado para la ACL que permitirá la regeneración in vivo, se requiere un modelo animal adecuado. En este estudio, la fabricación de un injerto de ingeniería biodegradable se describe, como es su implantación in vivo utilizando un modelo reproducible de la reconstrucción del LCA en una rata...

La rotura del ligamento cruzado anterior (LCA) es una de las más comunes las lesiones de ligamentos de la rodilla 1. Debido a la rotura de ligamento cruzado anterior de es incapaz de curar sin intervención quirúrgica, las limitaciones en las actividades de la vida diaria, así como la participación en deportes en coche más de 175.000 pacientes a someterse a cirugía cada año 2, con un costo estimado de mil millones de dólares al año 3. En la actualidad, ya sea autoinjerto o aloinjerto de tendón se utiliza para la reconstrucción del ligamento. Aunque altas tasas de éxito se puede lograr tanto con autoinjerto y aloinjerto de reemplazo, las complicaciones graves se asocian con estas opciones de reconstrucción 4. Tejido autoinjerto se asocia con morbilidad del sitio donante y se limita la oferta, sobre todo en casos de re-ruptura o lesiones múltiples ligamentosa. Por otro lado, el tejido de aloinjerto está vinculado con la integración retardada del injerto, la respuesta inflamatoria adversa, el riesgo infeccioso teórico, y supl limitadaly 5. Injertos no degradables sintéticos fueron desarrollados en los años 1970 y 1980, pero se vieron obstaculizados por la ruptura prematura del injerto, reacción de cuerpo extraño, osteolisis, y sinovitis 6. Como resultado de estas graves preocupaciones, actualmente no hay injertos sintéticos disponibles para uso clínico en los Estados Unidos. Debido a estas limitaciones con opciones de injerto existentes y a la evolución reciente de la biología, la ingeniería y la medicina regenerativa, ha habido un gran interés en una solución de ingeniería de tejidos para ACL injerto. Las estrategias de ingeniería de tejidos actuales emplean materiales biológicos y sintéticos degradables para permitir el crecimiento de tejido anfitrión evitando al mismo tiempo las limitaciones asociadas con la implantación de material sintético permanente 7. La policaprolactona (PCL) es un polímero biodegradable que es aprobado por la FDA para un número de aplicaciones médicas, incluyendo barrera de adhesión y un vendaje de heridas 8, que ha sido used en una amplia variedad de aplicaciones, incluyendo vascular, hueso, cartílago, nervios, piel, y la ingeniería de tejido esofágico 5,9-16. Biocompatibilidad favorable, relativamente larga in vivo la vida media, una resistencia mecánica adecuada, alta elasticidad y contribuyen a la popularidad de este polímero en la ingeniería de tejidos. En un modelo de roedor de la cicatrización de heridas, implantado PCL electrospun ha demostrado ser no inmunogénica y de integrarse en el tejido local sin reacciones adversas 13. Una imagen SEM de electrospun PCL se muestra en la Figura 1. Con actual de la FDA reguladora normas, la eficacia y la seguridad tanto en modelos animales pequeños y grandes que se requeriría para un PCL o cualquier otro injerto de LCA diseñado para moverse en ensayos clínicos en los Estados Unidos. Además, las condiciones in vivo a menudo pueden aumentar las propiedades de un injerto de tejido in vitro de ingeniería. Un modelo de rata de la reconstrucción del LCA autólogo con digitor flexorum tendón del se ha descrito anteriormente, en el que se rompió el ligamento cruzado anterior nativa, femoral y tibial túneles fueron perforados, y el injerto fue aprobada y se fija mediante sutura 17-22. En este artículo, vamos a describir una modificación de este modelo para la evaluación de los reemplazos de ACL ingeniería en lugar de para la reconstrucción a base de autoinjerto (Figura 2). Aunque existen muchos modelos animales para la ingeniería de tejido del ligamento, la rata es ventajoso en comparación con los modelos más grandes para un número de razones. Estas ventajas incluyen la cría y manejo más fácil, menos consideraciones éticas, y la reducción de 17,23 costo. Además, el modelo de rata se ha usado extensivamente como un modelo para la regeneración de tejido ortopédico, incluyendo el cartílago, tendón, y la ingeniería de tejido óseo 24. En particular, las ratas desnudos atímicos se eligieron debido a su falta de respuesta inmune mediada por células 25, permitiendo la implantación eventual of células del donante xenogeneicos en este modelo para mejorar aún más el injerto de ingeniería en el futuro. En este documento métodos, se describe la fabricación y la implantación quirúrgica de un acelular, injerto de polímero biodegradable en un modelo de rata atímicos de reconstrucción del LCA.

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	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:77px;">Catalog Number</td>
			<td style="width:317px;">Comments/Description</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Medical grade ester terminated poly (&#949;-caprolactone), granule form (MW = 110,000)</td>
			<td style="width:147px;">Lactel Absorbable Polymers</td>
			<td style="width:77px;"></td>
			<td style="width:317px;">Custom synthesized polymer to desired molecular weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>105228</td>
			<td style="width:317px;">Solvent for PCL polymer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">18G x 1 1/2&#34;bevel needle</td>
			<td style="width:147px;">BD Medical</td>
			<td>305196</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Remote Infuse/Withdraw Programmable Syringe Pump</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td style="width:77px;">702101</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">VersaLaser VLS2.30 Laser Engraver</td>
			<td style="width:147px;">Microgeo USA</td>
			<td style="width:77px;">VLS2.30</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Expanded Plasma Cleaner 115V</td>
			<td style="width:147px;">Harrick Plasma</td>
			<td style="width:77px;">PDC-001</td>
			<td style="width:317px;">Plasma etch just prior to collagen coating for surface modification</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">PureCol&#160; Collagen Standard Solution, 3 mg/ml</td>
			<td style="width:147px;">Advanced Biomatrix</td>
			<td style="width:77px;">5015-A</td>
			<td style="width:317px;">Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1N NaOH 1:9 in 1x PBS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Suture, 5-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1086471</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture, 4-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">6540072</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Sharp-pointed Dissecting Scissors (Straight; 4.5 inch)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:77px;">8940</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Buphrenorphine hydrochloride</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:77px;">B9275</td>
			<td style="width:317px;">Use 0.03 mg/kg for both intra- and post-operatively for pain control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Ampicillin, injectable</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1185678</td>
			<td style="width:317px;">Use 25 mg/kg subcutaneously during the procedure</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">K-wire, 1.6 mm</td>
			<td style="width:147px;">Spectrum Surgical</td>
			<td style="width:77px;">SI040062</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Keith Needle, Straight 1 1/2&#34;</td>
			<td style="width:147px;">Delasco Dermatology Lab &#38; Supply</td>
			<td style="width:77px;">KE-112</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Immunocal Decalcifying Solution</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>NC9491030</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td>
			<td style="width:147px;">US Dental Depot</td>
			<td style="width:77px;">OPTICRYL 100410&#160;</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:347px;">Instron Model 5564 Tensile Testing Machine</td>
			<td style="width:147px;">Instron</td>
			<td style="width:77px;">5564</td>
			<td style="width:317px;">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.

En nuestra experiencia de 92 cirugías de rata por un solo cirujano, el tiempo medio operatorio desde la incisión hasta la finalización de la herida fue de 16,9 min, con una desviación estándar de 4,7 min. En el momento del sacrificio, ratas pesaban 356 ± 23 g. Todas las ratas toleraron la cirugía bien, y no experimentaron complicaciones. Inmediatamente después de la cirugía, se observaron las ratas para soportar el peso sobre la extremidad operativa, pero mostraron una leve cojera. Una semana después de la ope...

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.

Atímicos Rata Modelo de Evaluación de Ingeniería de ligamento cruzado anterior injertos

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

Research

JoVE Journal

Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Metodología elastografía por resonancia magnética para la Evaluación de los tejidos Diseñado crecimiento Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Investigación

Bioingeniería

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

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

Recuperación Quirúrgica, aislamiento y<em&gt; In vitro</em&gt; Ampliación del cruzado anterior humana derivados de las células del ligamento para aplicaciones de ingeniería de tejidos

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

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Técnica quirúrgica para la implantación de la ingeniería tisular injertos vasculares y posterior<em&gt; En Vivo</em&gt; Monitoreo

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Generación y Acrecentamiento de buques de ingeniería de tejidos en un modelo de ratón

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

Producción, Caracterización y posibles usos de un Humano esofágica mucosas Modelo ingeniería tisular 3D

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Modelo de polarizado Neural tisular basada Silk-colágeno-Engineered 3D

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Aplicaciones de la<em&gt; En Vivo</em&gt; Pruebas funcionales de la rata tibial anterior para la evaluación de la ingeniería tisular de reparación del músculo esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

El escalado de Engineered injertos vasculares Usando 3D Impreso guías y el método de anillo de apilamiento

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

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

Tratamiento de las estructuras de ligamentos con suero acondicionado con ejercicio: Un modelo de ingeniería de tejidos traslacional

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

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Microbioreactor uso de alto rendimiento automático para la producción de IgG1 modelo en células CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...