The authors would like to acknowledge Stanford Department of Orthopaedic Surgery and Stanford Coulter Translational Seed Grant for funding. J.H.L. would like to thank National Science Foundation Graduate Fellowship and DARE Doctoral Fellowship for support.

Todos los experimentos se realizaron de acuerdo con las directrices de los sujetos de Stanford Universidad Humanos y aprobados protocolo Junta de Revisión Institucional. 1. Articular condrocitos aislamiento <ol><li> Obtener cartílago de tejidos descartados durante la artroplastia total de rodilla y diseccionar lo descrito previamente 8. </li><li> Visualmente seleccionar los cóndilos femorales medial o lateral de muestras de cartílago para superficies lisas y hacer la incisión en la superficie del cartílago a través de un escalpelo para eliminar 4-5 mm biopsias de cartílago sin afectar el hueso subcondral. </li><li> Aislar los condrocitos de la matriz extracelular por la disociación enzimática del tejido con tratamiento O / N con colagenasa purificada. Use 1: 1 relación de la colagenasa II (125 U / ml) y la colagenasa IV (160 U / ml) en medio de crecimiento de condrocitos a 37 ° C. </li><li> El día siguiente, filtra el tejido digerido que contiene las células disociadas a través de una 70mm poro tamaño de malla de nylon. De lavado dos veces con 20 ml de DMEM / F12 y centrifugar a 600 xg durante 5 min. </li><li> Placa de los condrocitos aislados a una densidad de 1 x 10 6 células / 60 mm placa de cultivo después de re-suspensión en DMEM / F12 suplementado con 10% suero bovino fetal (FBS), 25 mg / ml de ascorbato, 2 mM L-glutamina, antimicrobianos (100 U / ml de penicilina, 100 mg / ml de estreptomicina y 0,25 mg / ml de Fungizone). Mantener las placas a 37 ° C y mantener los cultivos de condrocitos primarios como una monocapa de alta densidad antes de la encapsulación en hidrogeles biomiméticos. </li></ol> 2. Biomimetic hidrogel Fabrication NOTA: Este método permite la síntesis de un hidrogel biomimético que contiene poli (etilenglicol diacrilato) (PEGDA, MW 5.000 g / mol), sulfato de condroitina-metacrilato (CS-MA) en DPBS. La adición de fotoiniciador permite reticulación fotoactivado. La composición de hidrogel final contiene 7% de pesajet / volumen (w / v) de PEGDA, 3% w / v de CS-MA, y 0,05% w / v de fotoiniciador. <ol><li> Sintetizar CS-MA por la funcionalización del polímero con grupos metacrilato CS. <ol><li> Preparar la solución tamponada de 50 mM de ácido 2-morfolinoetanosulfónico (MES) y cloruro de 0,5 M de sodio (NaCl) disolviendo 1,952 g de MES y 5,84 g de NaCl en 200 ml de agua desionizada (DiH 2 O). Disolver 5 g de sal sódica de sulfato de condroitina en la solución tamponada. </li><li> Añadir 0,532 g (4,62 mmol) de N-hidroxisuccinimida (NHS) y 1,771 g (9,24 mmol) de 1-etil-3- (3-dimetilaminopropil) carbodiimida (EDC) (relación molar de NHS: EDC = 1: 2) para la solución y se agita durante 5 min. </li><li> Añadir 0,765 g (4,62 mmol) de metacrilato de 2-aminoetilo (AEMA) (relación molar de NHS: EDC: AEMA = 1: 2: 1) a la solución y mantener la reacción a TA durante 24 h. </li><li> Se purifica la mezcla mediante diálisis frente a agua desionizada (DIH 2 O) durante 4 días utilizando un tubo de diálisis (12-14 kDa MWCO). </li><li> Se filtra la purified solución a través de un filtro de 0,22 micras y colocar los tubos abiertos que contienen la solución en el desecador de vacío y O / N. Asegúrese de que todos los tubos están protegidos de la luz. Guarde el polímero disuelto a -20 ° C protegido de la luz y la humedad. </li></ol></li></ol> 3. La encapsulación de la célula <ol><li> Autoclave las películas y cilíndricos varillas de PCR (para la perforación de los hidrogeles 3D de células con carga) y esterilizar el molde de gel cilíndrica medida por inmersión en 0,2 micras filtrada etanol al 70% en el tejido campana de cultivo bajo luz UV. </li><li> Sellar el fondo del molde con la película de gel PCR en autoclave sin burbujas o huecos para evitar fugas de aire. Guárdelo en una placa estéril 150 mm. </li><li> El día antes de la encapsulación de células en hidrogel biomimético, disociar la monocapa de condrocitos de alta densidad con O / N en el tratamiento de colagenasa II y solución de colagenasa IV. Utilice 125 U / ml para la colagenasa II y 160 U / ml para la colagenasa VI en el crecimiento de condrocitos cada mediosa 37 ° C. </li><li> Tomar un tubo de 50 ml y añadir 5% en peso / volumen (w / v) (diacrilato de etilenglicol) poli (PEGDA, MW = 5000 g / mol), 3% w / v sulfato de condroitina-metacrilato (CS-MA), y 0,05% w / v fotoiniciador en DPBS. Vortex el tubo para mezclar la solución y mantener a un lado. Proteja el tubo de la luz. </li><li> Recoger las células disociadas en un tubo Falcon 50 ml, contar y centrifugar a 460 xg durante 5 min. Aspirar todos los medios de comunicación desde el tubo de células y añadir el material de gel mixto anterior a las células volver a suspender a una densidad de 15 × 10 6 células / ml. Mezclar 30 veces a fondo y evitar la formación de burbujas de aire. </li><li> Pipeta 72 l de la suspensión de células de hidrogel o hidrogel solos en el molde de gel cilíndrica a medida e inducir la gelificación través de la exposición a la luz UV (365 nm de longitud de onda) a 3 mW / m 2 durante 5 min. Prepara un poco de solución de hidrogel sin contenido celular. Utilice estas construcciones como controles negativos para el futuro pruebas bioquímicas y mecánicas. Measure la intensidad de la luz UV con un medidor de intensidad de los rayos UV. Para ajustar la intensidad, cambiar la distancia entre la fuente de luz UV y el andamio de hidrogel durante la gelificación. NOTA: Dado que el hidrogel contiene CS, la contribución acelular se resta del contenido de GAG ​​bioquímica de los hidrogeles de células cargado para representar sólo el GAG celular. </li><li> Utilice un bisturí para cortar la película para fácil de despegar y retire con cuidado. Con la ayuda de las varillas cilíndricas, empujar hacia fuera el gel en una placa de 6 pocillos con 5 ml de DPBS estériles para lavar gel no polimerizado y las células sueltas. </li><li> Cultura los hidrogeles cargados de células en placas de 24 pocillos que contenían 1,5 ml de medio de crecimiento de condrocitos en cada pocillo. Use una espátula desechable para transferir los hidrogeles lavadas en el pozo. </li><li> Evaluar la viabilidad celular por muertos viven tinción 24 horas después de la encapsulación utilizando un kit de viabilidad / citotoxicidad. Cultura de los hidrogeles de células cargadas y los hidrogeles vacíos para 3-6 semanas cambiantes de gro condrocitos frescaswth medios cada 2 días antes de la cosecha para los análisis. </li></ol> 4. RNA de extracción y análisis de expresión de genes <ol><li> Aspirar con cuidado los medios de comunicación y poner estéril PBS 1X en el teléfono cargado, así como los hidrogeles de control vacías. </li><li> Con la ayuda de una espátula estéril, transferir cada hidrogel a un tubo Eppendorf de 1,5 ml limpio y añadir 250 l de Tri-reactivo a cada tubo. Siga las instrucciones del fabricante para aislar ARN. </li><li> Después de aislamiento de ARN y cuantificación uso 1 g de la misma de cada muestra y realizar una transcripción inversa en ADNc utilizando la alta capacidad de cDNA Transcription Kit inversa. </li><li> Para la PCR cuantitativa TaqMan Arrays utilizar Expresión Génica para el examen de la expresión de los genes de interés. Normalizar los niveles de expresión génica internamente para GAPDH. </li><li> Para las condiciones de PCR incluir una incubación de 2 min a 50 ° C para inactivar amplicones anteriores con uracil-DNA glycosylase, seguido por un 1060; min de incubación a 95 ° C para activar la Taq polimerasa. A continuación, realice ciclos cuarenta de PCR, que consta de 15 segundos a 95 ° C y 1 min a 60 ° C. </li></ol> 5. Los análisis bioquímicos <ol><li> Para cada constructos de células hidrogel cuantificar el ADN y glicosaminoglicanos sulfatados (sGAG) la producción de la siguiente manera. </li><li> Aspirar con cuidado los medios de comunicación y poner estéril PBS 1X en el teléfono cargado, así como los hidrogeles de control vacías. Pesar un tubo vacío y poner el hidrogel en el interior después de secante en papel de seda y registrar el peso o el peso húmedo de los hidrogeles. </li><li> Congelar cada hidrogel a -20 ° C en un tubo eppendorf de 1,5 ml por separado para la digestión enzimática y más tarde utilizar una parte alícuota del producto digerido para ejecutar ensayo de Picogreen (para ADN) y el ensayo de azul de metileno-Dimetil (por GAG). </li><li> Preparar la solución de papainase para la digestión enzimática de los hidrogeles. <ol><li> En primer lugar preparar el tampón PBE pesando 7,1 g de sodio phosphate dibásico (Na 2 HPO 4) y 1,6 g de sal disódica del ácido etilendiaminotetraacético (EDTA-Na 2). Disolver en 500 ml de dH 2 O, ajustar el pH a 6,5 y filtrar la solución purificada a través de un filtro de 0,22 mm. </li><li> Disolver 0,035 g de L-cisteína en 20 ml de tampón de PBE. Se filtra la solución y 100 l de enzima papaína estéril. </li></ol></li><li> Cuando esté listo para realizar los ensayos, tomar los tubos de -20 ° C y añadir 300 l de la solución de papaína listo para los hidrogeles congelados. Machacar el gel con un mortero y se mezcla bien con la mano del mortero motor. </li><li> Llevar el volumen a 500 l con solución de papaína adicional. Realice el mismo procedimiento para los geles de control. Se incuba a 60 ° C durante 16 horas 3,9. La solución que contiene el hidrogel digerido por lo tanto se puede utilizar para la cuantificación de ADN y GAG. Mida el contenido de ADN utilizando el ensayo Picogreen con el ADN del fago Lambda de serie siguiente pr del fabricanteotocol. </li><li> Cuantificar el contenido-GAG sulfatados utilizando el azul de 1,9-dimetilmetileno (DMMB) ensayo de unión de tinte con sulfato de condroitina tiburón de serie 10. Determinar el contenido de GAG ​​dividiendo la cantidad de GAG ​​para el peso húmedo correspondiente. </li></ol> 6. Pruebas Mecánicas <ol><li> Realizar ensayos de compresión no confinada utilizando un sistema de ensayo Instron 5944 equipado con una célula de carga de 10 N. <ol><li> Después de los días deseados de cultivo in vitro, realice la prueba de compresión en el cadalso de células hidrogel y los hidrogeles de control acelular. </li><li> Sumergir las muestras en un baño de PBS a RT y comprimir a una velocidad de 1% de deformación / seg a una cepa máximo de 15% 11,12 ya que el esfuerzo fisiológico experimentado por tejido de cartílago bajo condición de carga ha sido informado de que el 10-20% 13,14. </li></ol></li><li> Crear estrés vs. curvas de tensión y ajuste de la curva utilizando una ecuación polinómica de tercer orden. Determinar las compressive módulo tangente de la curva de ecuación de ajuste a valores de deformación de 15%. </li></ol>

<ol>
	<li>Roberts, S., Menage, J., Sandell, L. J., Evans, E. H., Richardson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+collagen+types+I+and+II+and+procollagen+IIA+in+human+cartilage+repair+tissue+following+autologous+chondrocyte+implantation.">Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation.</a> Knee. 16, 398-404 (2009).</li><li>Smeriglio, P., 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=Comparative+Potential+of+Juvenile+and+Adult+Human+Articular+Chondrocytes+for+Cartilage+Tissue+Formation+in+Three-Dimensional+Biomimetic+Hydrogels.">Comparative Potential of Juvenile and Adult Human Articular Chondrocytes for Cartilage Tissue Formation in Three-Dimensional Biomimetic Hydrogels.</a> Tissue engineering. Part A. , (2014).</li><li>Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+catalyze+cartilage+formation+by+neonatal+articular+chondrocytes+in+3D+biomimetic+hydrogels.">Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels.</a> Scientific reports. 3, 3553 (2013).</li><li>Taipale, J., Keski-Oja, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+in+the+extracellular+matrix.">Growth factors in the extracellular matrix.</a> FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 11, 51-59 (1997).</li><li>Varghese, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroitin+sulfate+based+niches+for+chondrogenic+differentiation+of+mesenchymal+stem+cells.">Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells.</a> Matrix Biol. 27, 12-21 (2008).</li><li>Park, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+matrix+stiffness+on+the+differentiation+of+mesenchymal+stem+cells+in+response+to+TGF-beta.">The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-beta.</a> Biomaterials. 32, 3921-3930 (2011).</li><li>Wang, T., Lai, J. H., Han, L. H., Tong, X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+Differentiation+of+Adipose-Derived+Stromal+Cells+in+Combinatorial+Hydrogels+Containing+Cartilage+Matrix+Proteins+with+Decoupled+Mechanical+Stiffness.">Chondrogenic Differentiation of Adipose-Derived Stromal Cells in Combinatorial Hydrogels Containing Cartilage Matrix Proteins with Decoupled Mechanical Stiffness.</a> Tissue engineering. Part A. , (2014).</li><li>Smith, R. 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=Effects+of+intermittent+hydrostatic+pressure+and+BMP-2+on+osteoarthritic+human+chondrocyte+metabolism+in+vitro.">Effects of intermittent hydrostatic pressure and BMP-2 on osteoarthritic human chondrocyte metabolism in vitro.</a> J Orthop Res. 29, 361-368 (2011).</li><li>Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., Kimura, J. H., Hunziker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocytes+in+agarose+culture+synthesize+a+mechanically+functional+extracellular+matrix.">Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix.</a> J Orthop Res. 10, 745-758 (1992).</li><li>Farndale, R. W., Buttle, D. J., Barrett, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+quantitation+and+discrimination+of+sulphated+glycosaminoglycans+by+use+of+dimethylmethylene+blue.">Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue.</a> Biochim Biophys Acta. 883, 173-177 (1986).</li><li>Lai, J. H., Levenston, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meniscus+and+cartilage+exhibit+distinct+intra-tissue+strain+distributions+under+unconfined+compression.">Meniscus and cartilage exhibit distinct intra-tissue strain distributions under unconfined compression.</a> Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society. 18, 1291-1299 (2010).</li><li>Li, L. P., Buschmann, M. D., Shirazi-Adl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-rate+dependent+stiffness+of+articular+cartilage+in+unconfined+compression.">Strain-rate dependent stiffness of articular cartilage in unconfined compression.</a> Journal of biomechanical engineering. 125, 161-168 (2003).</li><li>Armstrong, C. G., Bahrani, A. S., Gardner, D. L. <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+measurement+of+articular+cartilage+deformations+in+the+intact+human+hip+joint+under+load.">In vitro measurement of articular cartilage deformations in the intact human hip joint under load.</a> The Journal of bone and joint surgery. American. 61, 744-755 (1979).</li><li>Macirowski, T., Tepic, S., Mann, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+stresses+in+the+human+hip+joint.">Cartilage stresses in the human hip joint.</a> Journal of biomechanical engineering. 116, 10-18 (1994).</li><li>Benya, P. D., Shaffer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiated+chondrocytes+reexpress+the+differentiated+collagen+phenotype+when+cultured+in+agarose+gels.">Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels.</a> Cell. 30, 215-224 (1982).</li><li>Buckwalter, J. A., Mankin, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+cartilage:+tissue+design+and+chondrocyte-matrix+interactions.">Articular cartilage: tissue design and chondrocyte-matrix interactions.</a> Instructional course lectures. 47, 477-486 (1998).</li><li>Wang, D. 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=Multifunctional+chondroitin+sulphate+for+cartilage+tissue-biomaterial+integration.">Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration.</a> Nat Mater. 6, 385-392 (2007).</li><li>Simson, J. A., Strehin, I. A., Allen, B. W., Elisseeff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonding+and+fusion+of+meniscus+fibrocartilage+using+a+novel+chondroitin+sulfate+bone+marrow+tissue+adhesive.">Bonding and fusion of meniscus fibrocartilage using a novel chondroitin sulfate bone marrow tissue adhesive.</a> Tissue engineering. Part A. 19, 1843-1851 (2013).</li></ol>

The authors have nothing to disclose.

Como se informó en este protocolo, los hidrogeles 3D son capaces de mantener el fenotipo de los condrocitos en la cultura, evitando el proceso de desdiferenciación celular en células fibrocartílago usualmente encontrados con cultivos monocapa 15. Por otra parte, cultivos a largo plazo de la construcción de hidrogel chondrocytes- revelaron un entorno favorable que mantiene las características intrínsecas de células asociadas con la edad y la enfermedad. El uso de un hidrogel biomimético 3D tiene varias ventajas. En primer lugar, la inclusión de sulfato de condroitina (CS), un componente principal encontrado en el cartílago articular, permiten a las células para degradar la matriz de hidrogel mediante la secreción de condroitinasa y fijar en el cartílago recién sintetizado extra-celular de matriz 5, 16. Además, CS ha demostrado que tienen propiedades anti-inflamatorias en la articulación artrítica. El hidrogel biomimético también puede ser utilizado como un material de andamiaje para la entrega celular en la reparación del cartílago, y puede ser modificado químicamentepara facilitar una mejor integración 17,18 biomaterial-tejido. El uso de los hidrogeles PEG-CS permite cultivos a largo plazo de los condrocitos y la evaluación de las propiedades bioquímicas y mecánicas. Aquí mostramos cómo esta plataforma puede ser útil para los análisis comparativos de diversas fuentes de condrocitos diferenciados con el fin de definir el tipo de célula óptima para la ingeniería de cartílago. Curiosamente, los condrocitos encapsulados en hidrogeles permanecen viables y proliferan según sus capacidades intrínsecas. Los soportes composición de hidrogel, de hecho, el crecimiento de juveniles y adultos condrocitos sanos como se muestra en la Figura 2. La composición y estructura de los hidrogeles descritos también promueve la formación de tejido de cartílago como se indica por la deposición de una matriz extracelular funcional evaluado por glicosaminoglicanos (GAG ) cuantificación. Una ventaja adicional es que las construcciones de condrocitos hidrogelse puede evaluar para las propiedades mecánicas del tejido de cartílago recién formado. Tenga en cuenta que la prueba de compresión no confinada se debe realizar en el hidrogel acelular para la comparación. Los hidrogeles, de hecho, tienen una rigidez intrínseca debido a la rigidez de los restos de CS. Esfuerzo de compresión no confinada de 5-20% (a una velocidad de deformación 1% / s) se puede aplicar para el ensayo mecánico del tejido cartilaginoso 11,12 ya que el esfuerzo fisiológico experimentado por tejido de cartílago bajo condición de carga se ha informado a 10-20 13,14%. La respuesta de ambas células cargadas y acelular hidrogeles a ensayos mecánicos se evaluó en el punto final de la cultura. En el ejemplo descrito anteriormente se observó una rigidez comparable de las construcciones que contienen adultos y juveniles condrocitos en contraste con la menor rigidez de las construcciones que contienen condrocitos OA. Tales propiedades mecánicas de la construcción de células-hidrogel permiten la evaluación de las propiedades funcionales de latejido formado dando un análisis en profundidad de la capacidad de maduración de las células. En conclusión, el uso de los hidrogeles biomiméticos 3D para estudiar el potencial de los diferentes población de condrocitos para generar tejido cartilaginoso puede ser ampliamente aplicada. Además de los estudios in vitro descritos aquí, el trasplante in vivo de los constructos de células cargado se puede prever para estudiar la maduración celular y el potencial regenerativo en el contexto fisiológico. Otras modificaciones de la plataforma de hidrogel con factores biomiméticos adicionales también se pueden prever para optimizar la proliferación de condrocitos y la maduración.

With its limited self-repair potential, human articular cartilage undergoes frequent irreversible damages. Extensive efforts are currently focused on the development of efficient cell-based approaches for treatment of articular cartilage injuries. The success of these cell-based therapies is highly dependent on the selection of an optimal cell source and the maintenance of its regenerative potential. Chondrocytes are a common cell source for cartilage repair, but they are limited in supply and can de-differentiate during in vitro expansion in 2D monolayer culture thereby limiting their generation of hyaline cartilage 1. 
The aim of this protocol is to establish a 3-dimensional hydrogel platform for an in vitro comparative study of human chondrocytes from different ages and disease state. Unlike conventional two-dimensional (2D) culture, three-dimensional (3D) hydrogels allow chondrocytes to maintain their morphology and phenotype and provides a physiologically relevant environment enabling chondrocytes to produce cartilage tissue 2,3. In addition to providing a 3D physical structure for chondrocyte culture, hydrogels mimic the function of native cartilage extracellular matrix (ECM). Specifically, the inclusion of chondroitin sulfate methacrylate provides a potential reservoir for secreted paracrine factors 4 and enables cell-mediated degradation and matrix turnover 5. Although many 3D hydrogel culture systems have been utilized widely in various studies including agarose and alginate gels, we have used a biomimetic 3D culture system that has some distinct advantages for chondrocyte culture. Chondroitin sulfate (CS) is an abundant component in articular cartilage and the PEG-CS hydrogels have been shown to maintain and even enhance chondrogenic phenotype and facilitate cell-mediated matrix degradation and turnover 2,5. In addition, the mechanical properties of the hydrogel scaffold can be easily modulated by changing concentration of PEG and hence can be utilized to further enhance the regeneration potential of chondrocytes or a related cell type 6,7. PEG/CSMA is also biocompatible and hence has the potential for a direct clinical application in cartilage defects for example. The limitation for this system is its complexity and the use of photopolymerization that can potentially affect cell viability as compared to simpler systems like agarose, however the advantages for the chondrocyte culture outweigh the potential limitations.
The 3D hydrogel culture is compatible with conventional assay for evaluation of cell phenotype (gene expression, protein immunostaining) and functional outcome (quantification of cartilage matrix production, mechanical testing). This favorable 3D environment was tested to compare the tissue regeneration potential of human chondrocytes from three different aged populations in long-term 3D cultures. 
The outcomes were evaluated via both phenotypic and functional assays. Juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogel culture. After 3 and 6 weeks, chondrogenic gene expression was upregulated in juvenile and adult chondrocytes but was downregulated in OA chondrocytes. Deposition of cartilage tissue components including aggrecan, type II collagen, and glycosaminoglycan (GAG) was high for juvenile and adult chondrocytes but not for OA chondrocytes. The compressive moduli of the resulting cartilage constructs also exhibited similar trends. In conclusion, both juvenile and adult chondrocytes exhibited chondrogenic and cartilage matrix disposition up to 6 weeks of 3D culture in hydrogels. In contrast, osteoarthritic chondrocytes revealed a loss of cartilage phenotype and minimal ability to generate robust cartilage.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>juvenile chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System )</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>adult chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System)</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(ethylene glycol diacrylate)</td>
			<td>Laysan Bio</td>
			<td>ACRL-PEG-ACRL-1000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>2-morpholinoethanesulfonic acid</td>
			<td>Sigma</td>
			<td>M5287</td>
			<td></td>
		</tr>
		<tr>
			<td>photoinitiator</td>
			<td>Irgacure&#160;</td>
			<td align="right">2959</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride&#160;</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>chondroitin sulfate sodium salt&#160;</td>
			<td>Sigma</td>
			<td>C9819</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide&#160;</td>
			<td>Sigma</td>
			<td align="right">130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride</td>
			<td>Sigma</td>
			<td>E1769</td>
			<td></td>
		</tr>
		<tr>
			<td>2-aminoethyl methacrylate</td>
			<td>Sigma</td>
			<td align="right">516155</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Spectrum Laboratories&#160;</td>
			<td align="right">132700</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 2</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 4</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004189</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>HyClone, Thermo Scientific&#160;</td>
			<td>SH3002301&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>live/dead assay</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/L3224">L3224</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Tri reagent</td>
			<td>Life Technologies</td>
			<td>AM9738</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT&#8482; PicoGreen&#174; dsDNA Assay Kit</td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/P11496">P11496</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic&#160;</td>
			<td>Sigma</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt&#160;</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C1276</td>
			<td></td>
		</tr>
		<tr>
			<td>1,9-dimethylmethylene blue&#160;</td>
			<td>Sigma</td>
			<td align="right">341088</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV light equipment - XX-15LW Bench Lamp, 365nm</td>
			<td>UVP</td>
			<td>95-0042-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Instron 5944 testing system&#160;</td>
			<td>Instron Corporation</td>
			<td>E5940</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Hidrogeles bioactivos que contienen PEG y restos CS (Figura 1) representan una plataforma ideal para la cultura y la maduración de los condrocitos articulares humanos 2,3,5,7. Los condrocitos de diferentes edades y estados de enfermedad pueden ser cultivadas con el método descrito y analizado por su fenotipo, la expresión génica y propiedades bioquímicas y mecánicas del tejido de cartílago generado. Tres muestras de condrocitos, juvenile- 6 meses, edad adulta 34 años y osteoarthritic- 74 años, se recogieron después de 3 semanas de cultivo en hidrogeles biomiméticos 3D. La expresión génica de los condrocitos analiza normal, tanto juveniles y adultos, mostró un aumento en la expresión de los genes COL6A1 y condrocitos Col2a1. Por el contrario, los condrocitos enfermas mostraron una disminución dramática en Col2a1 manteniendo al mismo tiempo la expresión de COL6A1 (Figura 2) que muestra una pérdida de fenotipo condrogénica a pesar de ser cultivadas en un medio ambiente favorable biomimético. Los hidrogeles de PEG-CS también sirven como un entorno dinámico para los condrocitos a proliferan, digieren la matriz pre-existente de PEG y CS y depositan su matriz pericelular y extracelular, los principales componentes del tejido de cartílago maduro. Dadas estas capacidades, la expansión de condrocitos después de 3 semanas de cultivo in vitro se puede estimar mediante la cuantificación de ADN con el tinte Picogreen. Análisis comparativo de los tres grupos de células muestran que la densidad celular de las poblaciones de juveniles y adultos se mantuvo sin cambios, mientras que los condrocitos OA mostraron una disminución dramática en comparación con los días 1 de la cultura. Una pérdida de contenido de ADN también se observó para la OA, pero no otros condrocitos que sugieren la posible muerte celular durante el largo plazo la cultura (Figura 3). La matriz secretada se cuantificó como contenido-GAG sulfatado mediante el ensayo de fijación de colorante DMMB en la etapa de punto final después de 3 semanas de las culturas. Contenido de GAG ​​se normaliza por el peso húmedo del hidrogel como recorded antes de la digestión enzimática y la contribución acelular se resta. Como se muestra en la Figura 4, los condrocitos depositan una cantidad significativa similar de GAG durante 3 semanas de cultivo en hidrogeles 3D. La presencia de un andamio física permite que la evaluación de las propiedades biomecánicas de las muestras a través de no confinado ensayos de compresión 2,3. Mientras que los hidrogeles acelulares se someten a una disminución en los módulos de compresión, los constructos de células Laden mantienen los módulos de compresión después de 3 semanas en cultivo (Figura 4). El fenotípica, análisis bioquímicos y biomecánicos descritos aquí son por lo tanto útiles para evaluar y comprender el potencial de las diferentes poblaciones de condrocitos para el cartílago ingeniería. <img alt="Figura 1" src="/files/ftp_upload/53085/53085fig1.jpg" /> Figura 1. PEG-CS hidrogeles biomiméticos para cul de condrocitos 3Dtura. Los condrocitos se resuspenden en una mezcla que contiene poli (etileno glicol diacrilato) (PEGDA) y sulfato de condroitina-metacrilato (CS-MA) y fundido en el molde de gel cilíndrica a medida. Después de la exposición UV, solidificaron geles se recogen de los moldes y la viabilidad celular se evalúa por tinción muertos en vivo 24 horas después de la encapsulación. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53085/53085fig2.jpg" /> Figura 2. Expresión de genes condrogénicas en condrocitos humanos cultivados en hidrogeles biomiméticos 3D. La expresión génica cuantitativa de marcadores de cartílago Col2a1 y COL6A1 por juveniles, adultos y OA condrocitos después de 3 semanas de cultivo en hidrogeles biomiméticos 3D. Los valores se normalizaron a nivel de la expresión génica en el día 1. Error bars representan media ± DE. * p &lt;0,05 como se determina por una prueba t de Student de dos colas. Modificado de Smeriglio et al. 2 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53085/53085fig3.jpg" /> Figura 3. contenido de ADN en los condrocitos humanos cultivados en hidrogeles biomiméticos 3D. Cuantificación de ADN por el ensayo Picogreen en juveniles, adultos y OA condrocitos después de 3 semanas de cultivo en hidrogeles biomiméticos 3D. Los valores se normalizaron a nivel de ADN en el día 1. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;br /&gt; Figura medición de contenido 4. GAG y prueba de compresión no confinada de condrocitos humanos en el día 1 y después de 3 semanas de cultivo en hidrogeles biomiméticos 3D. (A) GAG cuantificación mediante ensayo DMMB en condrocitos en el día 1 y después de 3 semanas de cultivo en hidrogeles biomiméticos 3D. Los valores se normalizan a mojar peso (ww) y se expresaron como mg / g. (B) módulo de compresión (kPa) de acelulares y celulares cargados hidrogeles en el día 1 y después de 3 semanas de cultivo en hidrogeles biomiméticos 3D. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D hidrogel andamios para articular condrocitos cultura y cartílago Generación

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

3d-hydrogel-scaffolds-for-articular-chondrocyte-culture-cartilage

Research

JoVE Journal

Bioengineering

19.8K vistas.  Stanford University. Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes. 

Video: 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Una simple gota colgante celular Cultura Protocolo para la generación de esferoides 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Investigación

Bioingeniería

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparación de fibrina Andamios 3D para Aplicaciones madre para cultivo celular

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Generación de células epiteliales de las vías respiratorias del ratón ESC-Obtenido usando Descelularizado pulmón andamios

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Un experimental y el Protocolo de elementos finitos para investigar el transporte de solutos neutros y cargadas a través de cartílago articular

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D agregación magnética Stem Cell y biorreactor de maduración para la regeneración del cartílago

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting de cartílago y piel análogos de tejido utilizando un pasivo novela mezcla de unidad técnica de Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Visualización en tiempo real y análisis de la lesión de condrocitos debido a cargas mecánicas en explantes de cartílago murino intacta

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Fabricación de andamios de Decellularized derivados de cartílago matriz

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D impreso celulosa poroso nanocompuesto hidrogel andamios

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...