Name: 3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation
Uploaded: 2015-10-07T14:00:00.000+00:00
Duration: 12 min 37 s
Description: Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

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><tbody><tr><td>juvenile chondrocytes (Clonetics Normal Human Chondrocyte Cell System )</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>adult chondrocytes (Clonetics 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&amp;nbsp;</td><td>2959</td><td></td></tr><tr><td>sodium chloride&amp;nbsp;</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>chondroitin sulfate sodium salt&amp;nbsp;</td><td>Sigma</td><td>C9819</td><td></td></tr><tr><td>N-hydroxysuccinimide&amp;nbsp;</td><td>Sigma</td><td>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>516155</td><td></td></tr><tr><td>dialysis tubing</td><td>Spectrum Laboratories&amp;nbsp;</td><td>132700</td><td></td></tr><tr><td>Collagenase 2</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004177</td><td></td></tr><tr><td>Collagenase 4</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004189</td><td></td></tr><tr><td>DMEM/F12 media</td><td>HyClone, Thermo Scientific&amp;nbsp;</td><td>SH3002301&amp;nbsp;</td><td></td></tr><tr><td>live/dead assay</td><td>Life Technologies</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/L3224&quot;&gt;L3224&lt;/a&gt;</td><td></td></tr><tr><td>Tri reagent</td><td>Life Technologies</td><td>AM9738</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Invitrogen</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/P11496&quot;&gt;P11496&lt;/a&gt;</td><td></td></tr><tr><td>Sodium phosphate dibasic&amp;nbsp;</td><td>Sigma</td><td>S3264</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>341088</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>UV light equipment - XX-15LW Bench Lamp, 365 nm</td><td>UVP</td><td>95-0042-07</td><td></td></tr><tr><td>Instron 5944 testing system&amp;nbsp;</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

20.0K 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

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

Preparación de hidrogeles inyectables basados en quitosano y su aplicación en cultivos celulares 3D

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Investigación

Química

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

El cartílago humano Tissue Fabrication Usando tridimensional Tecnología de impresión inyección de tinta

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Bioingeniería

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Las construcciones en capas de alginato: Una plataforma para el co-cultivo de poblaciones celulares heterogéneas

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Cultivo de células de mamífero en andamios tridimensionales péptido

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Un sistema 3D para los condrocitos articulares humanos cultivo de líquido sinovial

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Biología

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

La estimulación mecánica de condrocitos-agarosa hidrogeles

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

Creación de una articulación de rodilla en un chip para modelar enfermedades articulares y probar medicamentos

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Síntesis de hidrogeles de matriz extracelular de cartílago descelularizado

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Generación de esferoides a partir de varios condrocitos utilizando condiciones de baja adherencia por gravedad y mini-biorreactores caseros

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...