1. Aislamiento de condrocitos articulares primarios Cosecha 10-15 rebanadas completo de cartílago de espesor de las superficies articulares de las articulaciones de animales (por ejemplo, la articulación metacarpiana-falángica de vacas esqueléticamente maduros obtenidos de un matadero local). <ol><li> Colocar las rodajas de cartílago en un 100 mm placa de Petri y se incuba en 20 ml de 0,5% de proteasa en Ham F-12 (w / v) durante 2 horas a 37 ° C. Enjuague tres veces en medios de cultivo de Ham F-12, y se incuba con 20 ml de 0,15% de colagenasa A en los medios de cultivo de Ham F-12 durante la noche a 37 ° C. </li><li> Se filtra la suspensión celular a través de un filtro de malla 200 de pantalla en un plato limpio. Lavar el filtro con 5 ml de Ham F-12 y añadir al plato. Transferir la suspensión celular a un tubo cónico de 50 ml. Lavar la placa de Petri con 10 ml de Ham F-12 y añadir al tubo cónico. </li><li> Centrifugar el tubo a 600-800 rcf durante 6-8 min a temperatura ambiente. </li><li> Aspirar el sobrenadante y asegúrese de no molestar ªe pastilla de células y volver a suspender en 40 ml de medio de cultivo de Ham F-12. </li><li> Centrifugar el tubo a 600-800 rcf durante 6-8 min. </li><li> Repetir los pasos 1,4 y 1,5 veces más para un total de 3 veces. Antes de la centrifugación de la tercera vez, resuspender el precipitado a través de la agitación y obtener una alícuota de 500 l para el recuento celular. </li><li> Contar las células viables usando el método de exclusión con azul de tripano 9 con un hemocitómetro y un microscopio de luz invertida. </li><li> Aspirar el sobrenadante y volver a suspender el sedimento en un volumen de 12-F de Ham al doble de la densidad de siembra deseada (por ejemplo 20 x 10 6 células / ml para una concentración final de 10 x 10 6 células / ml después de la encapsulación). </li></ol> 2. Condrocito-agarosa hidrogel Encapsulación <ol><li> Calor 0,8 g autoclave Tipo VII de agarosa en 20 ml de PBS 1x estéril (pH 7,4) en una placa caliente ajustada a 120 ° C hasta que se disolvió. Una vez disuelto, reducir el calor a 60 ° C como temperaturas más altas will afectar la viabilidad celular. </li><li> En un tubo cónico de 50 ml, mezclar cuidadosamente volúmenes iguales de la suspensión celular con el doble de la concentración deseada de agarosa (por ejemplo, 4% de suspensión de agarosa para una final de 2% de hidrogel de agarosa). Evitar la creación de burbujas grandes, ya que pueden afectar a la viabilidad celular y la construcción de la vida de fatiga. </li><li> Cuidadosamente alícuota de 10 ml de la mezcla de condrocito-agarosa en un diámetro de 60 mm placa de Petri, evitando al mismo tiempo la creación de burbujas grandes. </li><li> Dejar reposar durante 30 minutos a temperatura ambiente hasta que gelificó. </li><li> Obtener el mayor número de condrocitos-agarosa muestras de hidrogel, según sea necesario (típicamente 20-30) usando un punzón de biopsia de 4 mm. </li><li> Mida y registre las dimensiones físicas (diámetro y altura) de muestras representativas utilizando un micrómetro. Las construcciones debe ser aproximadamente 5 mm de altura, descartar cualquier muestras obtenidas de una región donde la varianza altura de la muestra fue mayor que 2%. </li><li> Transferir las muestras a un nuevo 60 mm placa de Petri y suplementocon 10 ml de medio completo (20% por ejemplo, suero bovino fetal en medios de cultivo de Ham F-12 con 20 mM de HEPES, 100 g / ml de ácido ascórbico, y antibióticos / antimicóticos 2x). </li></ol> 3. La estimulación mecánica y radiomarcaje de condrocitos-agarosa hidrogeles <ol><li> Esterilizar en autoclave los componentes metálicos de la instalación de compresión. Esterilizar los componentes de plástico con 70% de etanol (durante la noche) y luz UV (por lo menos durante 15 min). </li><li> Para mantener las construcciones que se caiga, usando fórceps, lugar 12 anillos de plástico estériles de retención (con un diámetro interior mayor que el diámetro constructo) (n = 6, muestras y controles) en los pocillos de una placa de cultivo de 24 pocillos. </li><li> Utilizando pinzas, transferir cuidadosamente los hidrogeles de condrocitos-agarosa de la placa de Petri a la placa de 24-así, asegurando cada uno en un anillo de retención. </li><li> Transferir 400 l de medio completo (por ejemplo, 20% de FBS en los medios de cultivo de Ham F-12 con 20 mM de HEPES, 100 ug / ml de ácido ascórbico, y2x antibióticos / antimicóticos) a cada pocillo de muestra. </li><li> Montar la plataforma de compresión esterilizada (Figura 1) y fije las placas con tornillos de fijación. Una el equipo de perforación de compresión a la placa de cultivo de 24 pocillos. </li><li> Suelte y vuelva a sujetar-los tornillos de ajuste para establecer platina de hidrogel de contacto (cero estado de deformación). </li><li> Cargue la plataforma de compresión montado en un Mach-1 Testing System micromecánica. Plataforma segura para la etapa vertical mediante pasador de posicionamiento y bloqueo en su lugar con el tornillo de fijación. Retire la plantilla de interlineado. </li><li> Aplicar una carga de compresión dinámica en amplitud deseada (por ejemplo, 10% de amplitud de deformación sobre la base de la altura original de las muestras), la frecuencia (por ejemplo, 1 Hz) y la duración o el número de ciclos (por ejemplo, intervalos de tiempo de hasta 60 min). </li><li> Tras la finalización de la estimulación mecánica, sustituir la plantilla espaciamiento, afloje el tornillo de pasador de fijación localizar y eliminar la plataforma de compresión y la placa de cultivo de Mach-1 Testing System micromecánicos. </li&gt; <li> Radiomarcador hidrogel células constructos suplementando el medio con 5 Ci de isótopo deseado (por ejemplo, [3 H]-prolina etiqueta de proteínas para la síntesis de colágeno específico de condrocitos y [35 S]-azufre para la síntesis de proteoglicanos). </li><li> Incubar los constructos radiomarcados para 24 horas a 37 ° C, 5% CO 2 10. </li><li> Cosechar los constructos de hidrogel radiomarcados y enjuague 3 veces en 1X PBS (pH 7,4) para eliminar cualquier isótopo no incorporado. Digest muestras usando papaína (40 ug / ml en 20 mM de acetato de amonio, 1 mM de ácido ethyldiaminetetraacetic y 2 mM de ditiotreitol a 65 ° C durante 72 hr). </li><li> Ensayo para digerir el contenido de ADN mediante el ensayo PicoGreen tinte 11 y actividad incorporada isótopo, normalizado a contenido de ADN, por β-líquido de centelleo contando 12,13 digestión con papaína inmediatamente siguiente. </li></ol> Nota: La compra y venta de materiales radiactivos (isótopos y elementos de desechoque puedan haber estado en contacto con materiales radioactivos) debe seguir el institucional relevante (y / o gubernamentales) Políticas y procedimientos para la manipulación y eliminación de sustancias radiactivas. 4. Los resultados representativos Bovina condrocitos articulares-agarosa construcciones hidrogeles (2% de agarosa con 10 x 10 6 células / ml células encapsuladas) fueron estimuladas mecánicamente a una amplitud de 10% de deformación a la compresión a una frecuencia de 1 Hz durante 20 a 60 min (o 1.200 ciclos a 3.600 ) y se ensayó el contenido de ADN y la síntesis de matriz extracelular mediante la incorporación de radioisótopos. ADN y la síntesis de matriz extracelular se vio afectada de una manera dependiente de la dosis. Contenido de ADN mostraron una disminución significativa del 35% como resultado de 20 o 30 min de estimulación (p &lt;0,01), mientras que 60 min de estimulación no mostró ningún efecto (Figura 2). Cartílago específicos de la síntesis de colágeno y proteoglicanos (determinado por [3 H]-prolina y[35 S]-azufre incorporación, respectivamente) mostraron incrementos significativos de aproximadamente 60% ​​en respuesta a 20 o 30 min de estimulación (p &lt;0,01), con 60 min de estimulación que no presenta efecto observable (Figura 3). <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="Figura 1" /> . Figura 1 Esquema de la plataforma de compresión dinámica hecha a la medida para estimular condrocitos agarosa construcciones de la izquierda:. Plataforma ensamblada Derecha:. Despiece que muestra los componentes individuales. <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="Figura 2" /> Cambios Figura 2. En el contenido de ADN de condrocito-agarosa hidrogeles mecánicamente estimulados bajo una amplitud de deformación 10% de compresión a 1 Hz durante 20 a 60 min (media ± SEM, n = 6/grupo). Cambios Figura 3. En la síntesis de matriz extracelular (colágeno y proteoglicanos) de condrocito-agarosa hidrogeles mecánicamente estimulados bajo amplitud de la deformación a la compresión 10% a 1 Hz durante 20 a 60 minutos (media ± SEM, n = 6/grupo).

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<html> El método descrito para la aplicación de estímulos mecánicos controlados a la celda de semilla de agarosa hidrogeles permite la investigación directa en los efectos dinámicos de las fuerzas de compresión sobre el metabolismo de los condrocitos. El uso de la plataforma de pruebas de medida junto con los anillos de retención proporcionado restricción lateral para las construcciones para evitar problemas potenciales de la muestra de inflexión. El uso de platos de carga muerta ponderados fijados por los equi...

<table border="1"><tbody><tr><td> Nombre del reactivo o equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Ham F-12 </td><td> Thermo Fisher Scientific </td><td> SH3001002 </td><td></td></tr><tr><td> Colagenasa A </td><td> Sigma Aldrich Ltd. </td><td> C0130 </td><td></td></tr><tr><td> Proteasa </td><td> Sigma Aldrich Ltd. </td><td> P5147 </td><td></td></tr><tr><td> Suero Bovino Fetal </td><td> Sigma Aldrich Ltd. </td><td> F1051 </td><td></td></tr><tr><td> Ascorbato </td><td> Sigma Aldrich Ltd. </td><td> A4034 </td><td></td></tr><tr><td> Antibióticos / antimicóticos </td><td> Sigma Aldrich Ltd. </td><td> A5955 </td><td></td></tr><tr><td> HEPES </td><td> Bioshop Canada Ltd. </td><td> HEP001 </td><td></td></tr><tr><td> Trypan azul </td><td> Sigma Aldrich Ltd. </td><td> 93595 </td><td></td></tr><tr><td> Reichert Bright-Line Hemocitómetro </td><td> Hausser Scientific </td><td> 1490 </td><td></td></tr><tr><td> Quant-iT PicoGreen </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> La papaína de la papaya látex </td><td> Sigma Aldrich Ltd. </td><td> P3125 </td><td></td></tr><tr><td> Acetato de amonio </td><td> Sigma Aldrich Ltd. </td><td> A1542 </td><td></td></tr><tr><td> Ácido Ethyldiaminetetraacetic </td><td> Sigma Aldrich Ltd. </td><td> E9884 </td><td></td></tr><tr><td> DL-ditiotreitol </td><td> Sigma Aldrich Ltd. </td><td> 43819 </td><td></td></tr><tr><td> Bajo punto de fusión de agarosa, Tipo VII </td><td> Sigma Aldrich Ltd. </td><td> A9045 </td><td></td></tr> &lt;tr&gt; <td> Pantalla de malla (200) Filtrar </td><td> Sigma Aldrich Ltd. </td><td> S4145 </td><td></td></tr><tr><td> Mach-1 Tester micromecánico </td><td> Biomomentum Inc. </td><td> V500cs </td><td></td></tr><tr><td> Compresión Cargando Jig </td><td> Custom-construido </td><td></td><td> Producto similar podría ser suministrada por Biomomentum Inc. </td></tr><tr><td> Falcon 24 Placa de Cultura Bien </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> β-líquido contador de centelleo </td><td> Beckman Coulter </td><td> LS6500 </td><td></td></tr><tr><td> [3H] Proline </td><td> Perkin-Elmer </td><td> NET323005MC </td><td></td></tr><tr><td> [35S] Azufre </td><td> Perkin-Elmer </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

El cartílago articular sufre de una capacidad limitada de reparación deteriorados por el insulto mecánico o degradados por la enfermedad, tal como la osteoartritis. Para remediar esta deficiencia, varias intervenciones médicas se han desarrollado. Uno de tales métodos es para recubrir la zona dañada con cartílago de tejido de ingeniería, sin embargo, la ingeniería de tejidos típicamente carece de las propiedades bioquímicas y la durabilidad de cartílago nativo, cuestionando su supervivencia a largo plazo. Esto limita la aplicación de la ingeniería de tejido cartilaginoso a la reparación de pequeños defectos focales, basándose en el tejido circundante para proteger el material implantado. Para mejorar las propiedades del tejido desarrollado, la estimulación mecánica es un método popular utilizado para mejorar la síntesis de la matriz extracelular cartilaginosa, así como las propiedades mecánicas resultantes de la ingeniería tisular. La estimulación mecánica se aplica fuerzas al tejido construye análoga a las experimentadas in vivo. Estase basa en la premisa de que el entorno mecánico, en parte, regula el desarrollo y mantenimiento de 1,2 tejido nativo. La forma más comúnmente aplicado de la estimulación mecánica en la ingeniería de tejido de cartílago es la compresión dinámica a cepas fisiológicos de aproximadamente 5-20% a una frecuencia de 1 Hz a 1,3. Varios estudios han investigado los efectos de la compresión dinámica y han demostrado que tienen un efecto positivo sobre el metabolismo de los condrocitos y la biosíntesis, en última instancia, afectar a las propiedades funcionales del tejido 4-8 desarrollado. En este trabajo, se expone el método para estimular mecánicamente condrocitos agarosa construcciones de hidrogel bajo compresión dinámica y analizar los cambios en la biosíntesis a través de ensayos bioquímicos y radioisótopos. Este método también puede ser fácilmente modificado para evaluar cualquier cambio potencialmente inducidos en respuesta celular como resultado de estímulos mecánicos.

Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

La biosíntesis de la matriz extracelular cartilaginosa por los condrocitos pueden ser afectados por la aplicación de estímulos mecánicos. Este método describe la técnica de aplicación de dinámicas tensiones compresivas a los condrocitos encapsulados en construcciones en 3D y la evaluación de los cambios inducidos en el metabolismo de los condrocitos.

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 ...

mechanical-stimulation-of-chondrocyte-agarose-hydrogels

Research

JoVE Journal

Biology

11.6K vistas.  Queen's University. La biosíntesis de la matriz extracelular cartilaginosa por los condrocitos pueden ser afectados por la aplicación de estímulos mecánicos. Este método describe la técnica de aplicación de dinámicas tensiones compresivas a los condrocitos encapsulados en construcciones en 3D y la evaluación de los cambios inducidos en el metabolismo de los condrocitos.

Video: Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

La diferenciación condrogénica de inducción de las células madre derivadas de tejido adiposo por la gravedad centrífuga

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Investigación

Biología del Desarrollo

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Diseño de un Carga mecánica Biaxial biorreactor para Ingeniería de Tejidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Bioingeniería

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 ...

Química

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.

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 ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Síntesis de termogelificante poli (N-isopropilacrilamida) compuestos de sulfato de condroitina-injertado con alginato micropartículas de Ingeniería de Tejidos

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Gradiente de tensión Chip para estimulantes comportamientos celulares en hidrogel cargadas de celulares

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

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 ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Una plataforma microfluídica para estimular los condrocitos con compresión dinámica

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

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

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 ...