DOZ con el apoyo de una subvención concedida por La Fundación de Ginebra. SN fue apoyado por una beca Postdoctoral Fellowship de la Iniciativa de Ingeniería de Tejidos de Pittsburgh.

1. Aislamiento adiposo células madre derivadas de (ASC) Nota: Todos los procedimientos se realizaron a temperatura ambiente a menos que se indique lo contrario. <ol><li> Aislar adiposo perirrenal rata y epididimal y se lava con solución estéril de Hank sal tamponada (HBSS) que contenía 1% de suero fetal bovino (SFB) como se describió anteriormente 6. </li><li> Picar el tejido y transferir 1-2 g en 25 ml de HBSS conteniendo FBS al 1% en un tubo de 50 ml y se centrifuga a 500 g durante 8 minutos a temperatura ambiente. </li><li> Recoger la capa adiposa de libre flotación del tejido y la transferencia a 125 ml matraz Erlenmeyer y se trata con 25 ml de colagenasa tipo II (200 U / ml) en HBSS durante 45 min a 37 ° C en un agitador orbital (125 rpm). </li><li> Retirar con cuidado la fracción líquida (por debajo de aceite y la capa de tejido adiposo) y filtrar secuencialmente a través de un filtro de nylon de malla 100-micras y 70 micras. Centrifugar el filtrado a 500 g durante 10 min a temperatura ambiente, aspirar el supernatant y lavar el precipitado dos veces con 25 ml de HBSS. </li><li> Resuspender el sedimento celular en 50 ml de medio de crecimiento (Medio MesenPRO RS basal), complementado con Suplemento MesenPRO Crecimiento RS, antibiótico-antimicótico (100 U / ml de penicilina G, 100 ug / ml de sulfato de estreptomicina, y 0,25 g / ml de anfotericina B) , y 2 mM de L-glutamina y células pipeta en 2 matraces T75 (25 ml / matraz). </li><li> Cultura de la ASC en un 5% de CO 2 humidificado incubadora a 37 ° C (Paso 4.2 ASC se utilizan para todos los experimentos). </li></ol> 2. Preparación de microesferas de quitosano (CSM) Nota: Todos los procedimientos se realizaron a temperatura ambiente a menos que se indique lo contrario. <ol><li> CSM se preparan mediante un proceso de agua en aceite emulsificación junto con una técnica de coacervación iónico utilizando nuestra anterior protocolo 6. Emulsionar una solución acuosa de quitosano (6 ml de w / v 3% de quitosano en 0,5 M de ácido acético) en un 100 ml de una mezcla en fase de aceite consistente en sojapetróleo, n-octanol (01:02 v / v) y 5% de sorbitan mono-oleato (Span 80) emulsionante, utilizando sobrecarga (1700 rpm) y agitación magnética (1000 rpm) simultáneamente, en direcciones opuestas. Este método doble de mezclado asegura que las micelas formadas desde el principio antes de la reticulación se produce pueden permanecer en solución y no se depositan en el fondo. Además, la barra magnética agitadora ayudas en DE-agregación de quitosano durante la formación de micelas y la rigidización. </li><li> La mezcla se agita continuamente durante aproximadamente 1 hora hasta una estable de agua-en-aceite que se obtiene. Reticulación iónica se inicia con la adición de 1,5 ml de 1% w / v de hidróxido de potasio en n-octanol cada 15 minutos durante 4 horas (24 ml en total). </li><li> Después de la terminación de la reacción de reticulación, lentamente decantar la fase oleosa de la mezcla que contiene CSM y añadir inmediatamente las esferas a 100 ml de acetona. Se lavan las esferas con acetona hasta que la fase de aceite se elimina completamente. </li><li> Secar los campos recuperados en un desecador de vacío y analyze sin más trámite. El tamaño medio de partícula CSM, área de superficie por miligramo y la unidad de volumen cúbico se determinó usando el analizador de tamaño de partícula. </li><li> Para los experimentos subsiguientes, lavar CSM tres veces con agua estéril para eliminar las sales residuales y esterilizar con alcohol absoluto. </li></ol> 3. Determinación del número de grupos amino libres en CSM Nota: Todos los procedimientos se realizaron a temperatura ambiente a menos que se indique lo contrario. <ol><li> Determinar el número de grupos amino libres presentes en CSM después de la reticulación iónica mediante el trinitro bencenosulfónico (TNBS) ensayo del ácido de Bubins y Ofner 7. Incubar 5 mg de microesferas con 1 ml de solución al 0,5% TNBS en un tubo de vidrio de 50 ml durante 4 horas a 40 ° C y hidrolizar con la adición de 3 ml de HCl 6N a 60 ° C durante 2h. </li><li> Enfriar las muestras a temperatura ambiente, y extraer los TNBS libres mediante la adición de 5 ml de agua desionizada y 10 ml de etiléter. </li><li> Calentar una alícuota de 5 ml de la fase acuosa a 40 ° C en un baño de agua durante 15 minutos para evaporar cualquier éter residual, enfriar a temperatura ambiente, y se diluye con 15 ml de agua. </li><li> Se mide la absorbancia a 345 nm con un espectrofotómetro utilizando la solución de TNBS sin quitosano como en blanco y quitosano el utilizado para la preparación CSM para determinar el número total de grupos amino. Estimación del número de grupos amino libres de la CSM en relación con quitosano. </li></ol> 4. Cargando ASC en CSM Nota: Todos los procedimientos se realizaron a temperatura ambiente a menos que se indique lo contrario. <ol><li> Equilibrar 5 mg de CSM esterilizados de la sección 2.5 en HBSS estériles durante la noche y agregar a un niño de 8 micras de tamaño de poro de inserción placa de la membrana de cultivo (placa de 24 pocillos). </li><li> Después de la CSM se han asentado sobre la membrana, cuidadosamente aspire el HBSS y añadir 300 l de medio de crecimiento hacia el interior del inserto y 700 l de medio de crecimiento para ªe fuera del inserto. </li><li> Resuspender ASC a la concentración apropiada (1 x 10 4 a 4 x 10 4) en 200 l de medio de crecimiento y las semillas sobre el CSM el interior del inserto placa de cultivo. El volumen final de medio de cultivo dentro de la inserción, después de la siembra, es de 500 l. </li><li> Incubar el cabeza de serie ASC a CSM durante 24 horas en un 5% de CO 2 humidificado incubadora a 37 ° C. </li></ol> 5. La determinación del porcentaje de carga ASC y la viabilidad celular en el CSM Nota: Todos los procedimientos se realizaron a temperatura ambiente a menos que se indique lo contrario. <ol><li> Después de la incubación, recoger el CSM ASC-cargada en un tubo de microcentrífuga de 1,5 ml estéril sin alterar las células que han migrado a la membrana de inserción. </li><li> Eliminar el medio residual y añadir 250 l de medio de cultivo fresco al tubo. </li><li> A cada tubo se añaden 25 l de MTT [3 - (4,5-dimethylthiozole-2-il) -2,5-difeniltetrazolio]solución (5 mg / ml) y se incuba durante 4 horas en un 5% de CO 2 humidificado incubadora a 37 ° C. </li><li> Después de la incubación, se elimina el medio, añadir 250 l de dimetilsulfóxido, y vórtice de la mezcla durante 2-5 minutos para solubilizar el complejo formazano. </li><li> Centrifugar el CSM a 2700 x g durante 5 min, y determinar la absorbancia sobrenadante medido a 570 nm utilizando 630 nm como referencia. </li><li> Determinar el número de células asociada con el CSM en relación con el valor de absorbancia obtenido a partir de un número conocido de viable ASC. </li></ol> 6. Caracterización de ASC-CSM-Embedded gel de colágeno Nota: Todos los procedimientos se realizaron a temperatura ambiente a menos que se indique lo contrario. <ol><li> Mezcla ASC-cargado CSM (5 mg contiene ≈ 2 x 10 4 células) con colágeno tipo 1 (7,5 mg / ml) extraído de tendón de cola de rata según el método de Bornstein 8 y fibrilar después de ajustar el pH a 6,8 usando NaOH 2 N. </li&gt; <li> Agregue el fibrilada colágeno-ASC-CSM mezcla a una placa de 12 pocillos y se incuba durante 30 minutos a un 5% de CO 2 humidificado incubadora a 37 ° C. </li><li> Después de la fibrilación completa, incubar los geles de colágeno-ASC-CSM para un máximo de 14 días en un 5% de CO 2 humidificado incubadora a 37 ° C. </li><li> Lanzamiento y la migración de las células de CSM en el gel se observaron utilizando técnicas estándar de microscopía. </li></ol> 7. Los resultados representativos En el presente estudio, hemos desarrollado una estrategia para entregar in vitro las células madre a partir de microesferas de quitosano (CSM) en un gel de colágeno andamio. CSM poroso de tamaño uniforme (175-225 micras de diámetro) y la composición se prepara y se usa como portadores de células (Figura 2). Tras incubar ASC con el CSM, las células unida a una concentración de 2 x 10 4 cells/5mg de CSM. Las células eran capaces de difundir sobre la microesfera, mientras que se extiende filopodiosen las grietas porosas de la microesfera (Figura 3). Una vez que el CSM cargadas con células se mezclaron con el gel de colágeno, las células inmediatamente comenzaron a emigrar en los geles (Figura 4). <img alt="Tabla 1" src="/files/ftp_upload/3624/3624table1.jpg" /> Tabla 1. Ventajas biológicas para el uso del quitosano y el colágeno en un sistema de entrega vástago célula. <img alt="Figura 1" src="/files/ftp_upload/3624/3624fig1.jpg" /> Figura 1. Esquema que representa la estrategia general para el uso dual de la ASC-CSM andamios de colágeno cargadas. Figuras 2, 3 y 4 se anotan en el esquema para ayudar con la interpretación de las imágenes. <img alt="Figura 2" src="/files/ftp_upload/3624/3624fig2.jpg" /> Figura 2. Representación esquemática que representa el proceso de siembra de células madre en quitosano microesferas. El procESS consiste en co-cultivo de ASC con el CSM en un 8 - tamaño de micras de poro la cultura inserción de la placa. Después de 24 horas, las microesferas son retirados de la inserción y están listos para incrustar en una matriz de biomaterial. <img alt="Figura 3" src="/files/ftp_upload/3624/3624fig3.jpg" /> Figura 3. Caracterización morfológica de CSM-cargado con ASC. El panel A muestra una micrografía de luz de ASC-cargado CSM, mientras que el panel B muestra el mismo campo de visión superpuesta con una imagen obtenida por microscopía de fluorescencia confocal. ASC se precargado con calceína AM (verde). El panel C muestra una imagen de una imagen SEM de una microesfera sin carga, mientras que el panel D muestra las células cargadas en la microesfera (asteriscos). La imagen de TEM en panel E muestra una sección transversal de una microesfera descargada. Una multitud de poros y grietas se encuentran a lo largo de la microesfera. Panel F muestra una sección transversal de microesferas con células (flechas) unida y que se extiende filopodios en la CREvicios. Aumentos originales: A y B = 70X, C = 500x, D = 2.000 x, E y F = 2.500 x. <img alt="Figura 4" src="/files/ftp_upload/3624/3624fig4.jpg" /> Figura 4. Migración de ASC desde el CSM en el armazón tridimensional de colágeno. Los paneles A y B representan el CSM con células migrando de microesferas y en la matriz de colágeno en el día 3 (A, las flechas). El panel B muestra una cultura similar después de 12 días. Microscopía electrónica de transmisión (TEM) se representan en las imágenes C, D y E. Los asteriscos en C y D muestran una microesfera que ha sido seccionada con células que migran fuera de la microesfera (flechas). Mayor aumento de panel D se representa en el panel E, y muestra filopodios de células asociadas a las fibrillas de colágeno (recuadro). Magnificación original: A y B = 100 veces; C y D = 6.000 x, E = 20.000 x, inserción = 150.000 x. <img alt="Figura 5" src="/files/ftp_upload/3624/3624fig5.jpg" /> Figura 5.Esquema que muestra los usos grandes de CSM en la medicina regenerativa y la administración de fármacos.

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No hay conflictos de intereses financieros existen. Aviso legal Las opiniones o afirmaciones contenidas en este documento son las opiniones particulares de los autores y no deben interpretarse como oficial o que refleja las opiniones del Departamento de Defensa o el Gobierno de los EE.UU.. Los autores son empleados del gobierno de EE.UU., y este trabajo fue preparado como parte de sus funciones oficiales. Todo el trabajo fue apoyado por el Ejército de los EE.UU. de Investigación Médica y el Comando de Material. Este estudio se llevó a cabo en virtud de un protocolo revisado y aprobado por la Investigación Médica del Ejército de EE.UU. y de la Junta de Revisión Institucional de Material de comandos, y de acuerdo con el protocolo aprobado.

Un obstáculo importante en las células madre basado en la terapia es el desarrollo de métodos eficientes para la entrega de las células de las regiones indicadas para su reparación. Debido a la variabilidad paciente a paciente, el tipo de tejido, el tamaño de la lesión y profundidad; la metodología de las células madre con envío debe determinarse sobre una base de caso por caso. A pesar de incorporar las células madre dentro de una matriz y su entrega a la zona de la herida que parece ser un enfoque más lóg...

<table border="1" ><tbody><tr><td> Nombre del reactivo / equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Hanks BalancedSalt solución (HBSS) </td><td> Gibco </td><td> 14175 </td><td> Consumible </td></tr><tr><td> Suero Bovino Fetal </td><td> Hyclone </td><td> SH30071.03 </td><td> Consumible </td></tr><tr><td> La colagenasa tipo II </td><td> Sigma-Aldrich </td><td> C6685 </td><td> Consumible </td></tr><tr><td> 70-m de nylon de malla de filtro </td><td> BD Biosciences </td><td> 352350 </td><td> Consumible </td></tr><tr><td> 100-m de nylon de malla de filtro </td><td> BD Biosciences </td><td> 352360 </td><td> Consumible </td></tr><tr><td> El crecimiento medio del sistema MesenPRO </td><td> Invitrogen </td><td> 12746-012 </td><td> Consumible </td></tr><tr><td> L-glutamina </td><td> Gibco </td><td> 25030 </td><td> Consumible </td></tr><tr><td> T75 frasco de cultivo de tejidos </td><td> BD Biosciences </td><td> 137787 </td><td> Consumible </td></tr><tr><td> El quitosano </td><td> Sigma-Aldrich </td><td> 448869 </td><td> Consumible </td></tr><tr><td> Ácido Acético </td><td> Sigma-Aldrich </td><td> 320099 </td><td> Consumible </td></tr><tr><td> N-octanol </td><td> Acros Organics </td><td> 150630025 </td><td> Consumible </td></tr><tr><td> Sorbitan mono-oleato </td><td> Sigma-Aldrich </td><td> S6760 </td><td> Consumible </td></tr><tr><td> Hidróxido de potasio </td><td> Sigma-Aldrich </td><td> P1767 </td><td> Consumible </td></tr><tr><td> Acetona </td><td> Fisher Scientific </td><td> L-4859 </td><td> Consumible </td></tr><tr><td> Etanol </td><td> Sigma-Aldrich </td><td> 270741 </td><td> Consumible </td></tr><tr><td> Trinitro bencenosulfónico </td><td> Sigma-Aldrich </td><td> P2297 </td><td> Consumible </td></tr><tr><td> Ácido clorhídrico </td><td> Sigma-Aldrich </td><td> 320331 </td><td> Consumible </td></tr><tr><td> Éter etílico </td><td> Sigma-Aldrich </td><td> 472-484 </td><td> Consumible </td></tr><tr><td> 8 micras de tejido Inserta cultivo en placa </td><td> BD Biosciences </td><td> 353097 </td><td> Consumible </td></tr><tr><td> 1,5 ml Tubos de microcentrífuga </td><td> Pescador </td><td> 05-408-129 </td><td> Consumible </td></tr><tr><td> Reactivo MTT </td><td> Invitrogen </td><td> M6494 </td><td> Consumible </td></tr><tr><td> Dimetilsulfóxido </td><td> Sigma-Aldrich </td><td> D8779 </td><td> Consumible </td></tr><tr><td> Qtracker célula Etiquetado Kit (Q seguimiento de 655) </td><td> Sondas moleculares </td><td> Q2502PMP </td><td> Consumible </td></tr><tr><td> Colágeno tipo 1 </td><td> Travigen </td><td> 3447-020-01 </td><td> Consumible </td></tr><tr><td> Hidróxido de sodio </td><td> Sigma-Aldrich </td><td> S8045 </td><td> Consumible </td></tr><tr><td> 12 placas de cultivo tisular </td><td> BD Biosciences </td><td> 353043 </td><td> Consumible </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5417R </td><td> Equipo </td></tr><tr><td> Agitador orbital </td><td> New Brunswick Scienctific </td><td> C24 </td><td> Equipo </td&gt; </tr><tr><td> Incubador humidificado con Air-5% de CO 2 </td><td> Thermo Scientific </td><td> Modelo 370 </td><td> Equipo </td></tr><tr><td> Agitador de varillas </td><td> IKA </td><td> Visc6000 </td><td> Equipo </td></tr><tr><td> Agitador magnético </td><td> Corning </td><td> PC-210 </td><td> Equipo </td></tr><tr><td> Desecador de vacío </td><td> - </td><td> - </td><td> Equipo </td></tr><tr><td> Analizador de Tamaño de Partículas </td><td> Malvern </td><td> STP2000 Spraytec </td><td> Equipo </td></tr><tr><td> Baño de agua </td><td> Fisher Scientific </td><td> Isotemp210 </td><td> Equipo </td&gt; </tr><tr><td> Espectrofotómetro </td><td> Beckman </td><td> Beckman Coulter DU800UV/Visible Espectrofotómetro </td><td> Equipo </td></tr><tr><td> Vórtice </td><td> Diagger </td><td> 3030a </td><td> Equipo </td></tr><tr><td> Lector de microplacas </td><td> Molecular Devices </td><td> SpectraMax M2 </td><td> Equipo </td></tr><tr><td> Luz / microscopio de fluorescencia </td><td> Olimpo </td><td> IX71 </td><td> Equipo </td></tr><tr><td> Microscopio Confocal </td><td> Olimpo </td><td> FV-500 microscopio confocal de barrido láser </td><td> Equipo </td></tr><tr><td> Microscopio Electrónico de Barrido </td><td> Carl Zeiss MicroImaging </td><td> Leo 435 VP </td><td> Equipo </td></tr><tr><td> Microscopio Electrónico de Transmisión </td><td> JEOL </td><td> JEOL 1230 </td><td> Equipo </td></tr></tbody></table>

constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

Las células madre pluripotentes han demostrado ser extremadamente útil en el campo de la medicina regenerativa 1-3. Sin embargo, con el fin de utilizar eficazmente estas células para la regeneración de tejido, un número de variables deben ser tenidas en cuenta. Estas variables incluyen: el volumen total y el área superficial del sitio de implantación, las propiedades mecánicas de los tejidos y el microambiente del tejido, que incluye la cantidad de vascularización y los componentes de la matriz extracelular. Por lo tanto, los materiales que se utilizan para entregar estas células deben ser biocompatibles con una composición química definida mientras se mantiene una resistencia mecánica que imita el tejido huésped. Estos materiales también debe ser permeable al oxígeno y nutrientes para proporcionar un microambiente favorable para las células para sujetar y proliferar. El quitosano, un polisacárido catiónico con una excelente biocompatibilidad, puede ser fácilmente modificado químicamente y tiene una alta afinidad para unirse con in vivo macromolecules 4-5. El quitosano imita la porción glucosaminoglucano de la matriz extracelular, lo que le permite funcionar como un sustrato para la adhesión celular, la migración y proliferación. En este estudio se utilizan quitosano en forma de microesferas para entregar derivadas de tejido adiposo células madre (ASC) en una base de colágeno tridimensional andamio 6. Un ideal de célula a microesfera relación se determinó con respecto al tiempo de incubación y densidad de las células para alcanzar el número máximo de células que podrían ser cargados. Una vez ASC se siembran en las microesferas de quitosano (MCS), que están incorporados en un armazón de colágeno y pueden mantenerse en cultivo durante períodos prolongados. En resumen, este estudio proporciona un método para entregar, precisamente, las células madre dentro de un andamio de tres dimensiones biomaterial.

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Un obstáculo importante en las actuales terapias con células madre es determinar el método más eficaz para proporcionar a estas células a los tejidos del huésped. A continuación, describimos un método de entrega basado en quitosano que sea eficiente y simple en el planteamiento, al tiempo que permite derivadas de tejido adiposo de células madre para mantener su multipotencia.

La construcción de un hidrogel de colágeno para la entrega de la madre microesferas cargadas con células quitosano

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

constructing-collagen-hydrogel-for-delivery-stem-cell-loaded-chitosan

Research

JoVE Journal

Bioengineering

16.7K vistas.  United States Army Institute of Surgical Research. Un obstáculo importante en las actuales terapias con células madre es determinar el método más eficaz para proporcionar a estas células a los tejidos del huésped. A continuación, describimos un método de entrega basado en quitosano que sea eficiente y simple en el planteamiento, al tiempo que permite derivadas de tejido adiposo de células madre para mantener su multipotencia.

Video: Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

El colágeno organotípicos I del ensayo: Una plataforma maleable para evaluar el comportamiento celular en un contexto de 3 dimensiones

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Investigación

Bioingeniería

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Un método mejorado para la preparación de colágeno tipo I De Piel

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging desnaturalizadas colágeno Strands<em&gt; En vivo</em&gt; Y<em&gt; Ex vivo</em&gt; A través de Photo-desencadenada Hibridación de Caged colágeno Mimetic Péptidos

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Desarrollo de amelogenina-quitosano hidrogel para<em&gt; In Vitro</em&gt; Esmalte Recrecimiento con una interfaz Dense

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Vortex asistida Microscale Electroporador para entrega secuencial Molecular

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Recombinante colágeno péptido Microcarriers para la extensión celular y su posible uso como sistema de células en un biorreactor modelo

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Fabricación de un hidrogel de derivados de matriz extracelular renal corteza

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an injury leading to delayed wound healing. We selected doxycycline (DOX), as a potential drug of choice, due to its anti-bacterial properties along with its reported anti-inflammatory properties. The current study aims to formulate DOX loaded collagen-chitosan non-crosslinked (NCL) &amp; crosslinked (CL) scaffolds and evaluate their healing ability in diabetic conditions. The characterization result of scaffolds reveals that the DOX-CL scaffold holds ideal porosity, a low swelling &amp; degradation rate, and a sustained release of DOX compared to the DOX-NCL scaffold. The in vitro studies reveal that the DOX-CL scaffold was biocompatible and enhanced cell growth compared with CL scaffold treated and control groups. The anti-bacterial studies have shown that the DOX-CL scaffold was more effective than the CL scaffold against the most common bacteria found in DW. Using the streptozotocin and high-fat diet-induced DW model, a significantly (p&#8804;0.05) faster rate of wound contraction in the DOX-CL scaffold treated group was observed compared to those in CL scaffold treated and control groups. The use of the DOX-CL scaffold can prove to be a promising approach for local treatment for DWs.

The prepared DOX-CL scaffold satisfied the prerequisites of an ideal DW dressing in mechanical strength, porosity, water absorption, degradation rate, sustained release, anti-bacterial, biocompatibility, and anti-inflammatory properties, which are considered to be essential for the recovery of damaged tissue in DWs.

Andamio compuesto de colágeno-quitosano cargado de doxiciclina para la curación acelerada de heridas diabéticas

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Preparación y caracterización de biotinta de hidrogel biohíbrida 3D basada en grafeno para neuroingeniería periférica

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Fabricación de microgeles de quitosano-genipina de tamaño controlado y sin emulsión para aplicaciones de ingeniería de tejidos

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...