Este trabajo fue apoyado por la Beca de Investigación UWS.

1. Chitosan adhesiva Preparación <ol><li> El polvo de quitosano es soluble en solución de ácido acético; para la preparación de una solución madre de ácido acético (2% v / v), añadir 10 ml de ácido acético glacial a 490 ml de agua desionizada (DI-H 2 O). </li><li> Para la preparación de una solución madre de Rosa de Bengala (RB) (0,01% w / v) en ácido acético, pesan 5 mg de RB en un vial envuelto con papel de aluminio para evitar la fotodecoloración. Añadir 0,5 ml de DI-H 2 O para disolver el polvo a continuación, añadir 49,5 ml de solución de ácido acético acciones (que se describe en la sección 1.1). </li><li> Para la preparación de quitosano solución (1,7% w / v), pesan 0,85 g de polvo de quitosano (MW medio, 85% grado de acetilación) y añadir a 50 ml de solución de ácido acético RB. El control se preparó por disolución de quitosán en la solución de ácido acético sin RB. </li><li> Añadir un agitador magnético a la mezcla de quitosano y se agita a temperatura ambiente. Para asegurar la disolución completa de los componentes, se agita la quitosanaen ácido acético + RB durante dos semanas y el quitosano en ácido acético durante 5 días (control). Cuando quitosano se disuelve, el pH de la solución aumenta desde ~ 2,6 a ~ 3,9. Tenga en cuenta que RB es poco soluble en pH ácido y que requiere un aumento en el pH de la solución y un tiempo prolongado de agitación para disolver. </li><li> Centrifugar las soluciones a 3270 xg durante 1 hora y se recoge el sobrenadante usando, por ejemplo, una jeringa estéril de 10 ml. Asegúrese de no molestar a la impureza de pellets. Durante este paso, la solución se limpia de impurezas macroscópicas y la materia insoluble. </li><li> Se inyecta la solución (centrifuga) quitosano en una losa perpex plana utilizando una jeringa estéril. El espesor de la película seca depende de la relación entre la cantidad dispensada de la solución y el área de superficie sobre la que se extiende la solución. Asegúrese de que la solución está libre de burbujas de aire y se extendió uniformemente sobre la losa. Para obtener películas delgadas con un espesor de 13 micras ~, ~ inyectar 1 ml de solución de más de 12 cm 2 de superficieárea (dimensiones 3x4 cm ~, Figura 1A). </li><li> Cubrir la placa de plexiglás con una pantalla, de lámina de aluminio por ejemplo, para evitar la fotodecoloración de la solución. Asegúrese de que la ventilación de aire suficiente, se garantiza que se seque la solución bajo la pantalla. </li><li> Agregar la solución se seque a temperatura ambiente (25 ° C) y presión (1 atm) hasta que la película resultante es insoluble en agua y no se hincha macroscópicamente. Esto se consigue típicamente en 2 semanas. </li><li> Separe las películas cuidadosamente con una espátula delgada y plana: estas películas se pueden despegar fácilmente el bloque de metacrilato sin dañarlos. </li><li> Medir el espesor de las películas utilizando un micrómetro. </li><li> Cortar el adhesivo en tiras rectangulares con una tijera afilada y limpia y colocarlos entre las diapositivas de vidrio estériles para preservar su forma plana. Envuelva las diapositivas en papel de aluminio para blindaje de luz y almacenar a temperatura ambiente. Etiquetar los adhesivos con o sin RB como &quot;rosa adhesivo&quot; o &quot;ADHES quitosanoive &quot;, respectivamente. </li><li> La rosa películas adhesivas se pueden esterilizar utilizando etanol (90%) o con radiación gamma (0,1 kGy / h durante 24 h) 4,5. </li></ol> 2. Atenuación óptica adhesivo <ol><li> Fije el adhesivo de color de rosa en el interior de una cubeta de cuarzo. </li><li> Registrar su espectro atenuada en el intervalo de 400 a 800 nm utilizando un espectrofotómetro de doble haz 4. </li><li> Suponiendo que la validez de la ley de Beer, el cálculo de la longitud de atenuación del adhesivo de la siguiente manera: I = I 0 e-Ax donde 0 es la intensidad del haz incidente, 1 / A es la longitud de atenuación, y x es el espesor de la película. </li><li> Repetir los pasos desde 2,1 hasta 2,3 para el adhesivo de quitosano para servir como un control. </li></ol> 3 En la solicitud de Vitro:. Láser unión del tejido en el intestino <ol><li> Harvest intestino de ternera inmediatamente después de la eutanasia de animales y se almacena a -80 ° C. </li><li> Antes de su uso, descongelar y el hidrato de tincidencia sumergiéndolo en DI-H 2 O durante 10 min. </li><li> Bisecar el tejido en secciones (2 x 1 cm) usando una cuchilla # 10 bajo un microscopio de operaciones (x10). Mantenga la humedad de las secciones usando DI-H 2 O. </li><li> Lleve los tocones incisión muy juntos (de extremo a extremo). Limpie el exceso de agua de la superficie con puntas de algodón. </li><li> Colocar una tira de adhesivo rosa (10 x 6 mm) a través de la incisión en la capa serosa con microforceps asegurando un contacto completo con el intestino (Figura 2). </li><li> La adherencia del tejido del adhesivo rosa se ​​activa mediante un láser bombeado por diodo de estado sólido, que está acoplado a una fibra óptica multimodo (diámetro del núcleo de 200 micras) 4. </li><li> Establecer el nivel de potencia del láser de 180 mW e irradiar el adhesivo para ~ 6 min con un tamaño de punto del haz de ~ 5 mm, utilizando los parámetros listados en la Tabla 1. Terreno irradiar el adhesivo asegurar que cada punto se irradió durante ~ 5 segundos antes de pasar el haz al punto adyacente. Tsu procedimiento garantiza que el haz de láser explora toda la superficie de las varias veces adhesivas. </li><li> Para evaluar la resistencia de la unión de tejido, una muestra de la abrazadera a un tensiómetro de columna calibrada único (Instron, MA, EE.UU.), utilizando mordazas mecánicas. Mover las empuñaduras en 22 mm / min hasta que los dos muñones de tejido separadas 4. </li><li> Coloque un adhesivo de color de rosa en el tejido como se describe en el paso 3.5. No irradiar la muestra y medir la resistencia de la unión de tejido como se describe en el paso 3,8. Este ejemplo sirve como control. </li></ol> 4 En la solicitud Vivo:. Anastomosis del nervio tibial <ol><li> Sedate la rata Wistar con isoflurano / O 2 mezcla (4% durante la inducción, 2% en adelante) 5. Inyectar por vía subcutánea Buprenex (0,03 mg / kg) para proporcionar intra-operatoria actividad analgésica y aliviar el dolor o incomodidad posible. </li><li> El procedimiento quirúrgico siguiente debe realizarse bajo un microscopio de operaciones (x10). Hacer una piel oblicuoincisión de unos 3 cm de largo en la parte dorso-lateral del muslo derecho y exponer el nervio tibial con un enfoque de división de músculo a través de los músculos de los glúteos 2. </li><li> Parcialmente diseccionar y cortar la adventicia del nervio tibial utilizando micro-tijeras rectas y absorber el exceso de agua con una gasa estéril o puntas de algodón. </li><li> Cortar el nervio tibial con micro-tijeras. </li><li> Posicionar una tira estéril de quitosano (dimensiones 5 x 4 mm) por debajo del nervio tibial utilizando micro-pinza. </li><li> Aproximado de los muñones del nervio extremo a extremo con micro-pinza sobre la tira adhesiva rosa. </li><li> La tira se adhiere plenamente a la del nervio que forma un collar, que puede ayudar en la rotación del nervio durante la irradiación con láser de la tira. </li><li> Activar el adhesivo se elevó con el láser como se describe en los pasos 3,6) y 3,7). </li><li> Recortar el adhesivo redundante aumentó desde el collar del nervio operado. </li><li> Cierre los músculos y la piel, con cinco puntos de sutura 3-0 5. </li&gt; <li> Estándar postoperatorias procedimientos de recuperación se deben seguir incluyendo la inspección diaria de dehiscencia o formación de pus y la administración de analgésicos, si es necesario. </li></ol> 5. Los resultados representativos Las películas que se obtienen son de color rosa brillante, fino y tener una superficie lisa (figura 1). Ellos también son flexibles y se puede enrollar en tubos de pequeño diámetro sin lágrimas que causan o cualquier otro daño aparente (Figura 1B). El adhesivo rosa tiene dos picos de absorción a 530 y 562 nm, el láser verde es así fuertemente absorbida por el adhesivo y la longitud de atenuación correspondiente a 532 nm es de ~ 12 m (Figura 3). En contraste, las películas de quitosano sin RB atenúa débilmente el láser (1 / a ~ 162 micras), probablemente debido a la dispersión. Se desprende de la trama espectros que ninguna agregación significativa de RB ocurre en las películas. La rosa uniones adhesivas firmementeal intestino tras la irradiación con láser (Figura 2) lograr una carga máxima típica en el fracaso de 0,9 ± 0,4 N (n = 30). La fuerza adhesiva se estimó como la carga máxima dividida por el área de la superficie adhesiva, a saber, 15 ± 6 kPa (n = 30). El adhesivo no irradiado rosa unido significativamente menor en el tejido (2 ± 2 kPa, n = 30, prueba t no pareada p &lt;10 6). La anastomosis in-vivo del nervio tibial se logró con éxito después de la aplicación y la unión del adhesivo rosa en conjunto con el láser verde. Una semana después de la operación, el nervio operado estaba en la continuidad y la fuerza de reparación fue de 17 ± 9 kPa (n = 10). <img alt="Figura 1" src="/files/ftp_upload/4158/4158fig1.jpg" /> Figura 1. A) La solución RB-quitosano es fundido en un plano Perspex losa (espesor de la losa ~ 5 mm). Papel de gráfico se coloca debajo de la losa para facilitar el paso seco de fundición. La f insoluble en aguailm se obtuvieron dos semanas después de la fundición. B) El adhesivo rosa es flexible y puede ser enrollado en un cilindro pequeño. <img alt="Figura 2" src="/files/ftp_upload/4158/4158fig2.jpg" /> Figura 2. El adhesivo rosa está unido al intestino de ternera después de la irradiación láser. Irradiación uniforme se aplica sobre el adhesivo RB durante adhesión a tejidos; manchas sin embargo seleccionados se muestran en esta imagen para ilustrar el efecto foto-blanqueamiento del láser sobre el adhesivo. <img alt="Figura 3" src="/files/ftp_upload/4158/4158fig3.jpg" /> Figura 3. El espectro de absorción (en unidades arbitrarias) del adhesivo rosa muestra dos picos a 530 y 562 nm. El láser verde (λ = 532 nm) es por lo tanto fuertemente absorbida por el adhesivo durante la exposición al láser. El adhesivo de quitosano sin RB absorbe mal el haz de láser. <table border="1"><tbody><tr><td></td><td> n </td><td><strong&gt; Area (mm 2) </td><td> Potencia (W) </td><td> Tiempo (s) </td><td> Fluence (J / cm 2) </td><td> I (W / cm 2) </td><td> Max Load / Area (kPa) </td></tr><tr><td> Adhesivo + Laser </td><td> 30 </td><td> 60 ± 10 </td><td> 0,18 ± 0,03 </td><td> 365 ± 5 </td><td> ~ 110 </td><td> 0,9 ~ </td><td> 15 ± 1 </td></tr><tr><td> Adhesivo </td><td> 30 </td><td> 60 ± 10 </td><td> NA </td><td> NA </td><td> NA </td><td> NA </td><td> 2 ± 2 </td></tr></tbody></table> Tabla 1. Legend. n, número de muestra, área, superficie del adhesivo rosa (media ± error máximo); energía, potencia del láser (media ± error máximo); Tiempo, tiempo de irradiación (media ± error máximo); Fluence, fluencia del láser promedio, I, estimado irradiance; máximo de carga / área, carga máxima a fallo del tejido reparado dividido por el área de la superficie adhesiva, (media ± SE).

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No hay conflictos de interés declarado.

La rosa fabricación adhesivo se basa en un sencillo proceso en seco fundido, aunque la disolución de RB en pH ácido requiere agitación prolongada de la solución de quitosano. Es importante dejar que la solución se seque hasta que se convierte en una película insoluble en agua. Esto sucede cuando el contenido en peso de agua es de ~ 10% en la película seca 6. Películas insolubles se obtienen normalmente después de 2 semanas en seco de colada en condiciones estándar de temperatura y presión (~ 25 °...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Rosa de Bengala </td><td> Sigma-Aldrich </td><td> 632-69-9 </td><td></td></tr><tr><td> Quitosano (MW medio) </td><td> Sigma-Aldrich </td><td> 10318AJ </td><td></td></tr><tr><td> Ácido acético glacial </td><td> Sigma-Aldrich </td><td> 08050051 </td><td> 2% v / v en agua DI </td></tr><tr><td> Agitador magnético </td><td> Heidolph </td><td> MR Hei-Mix S </td><td></td></tr><tr><td> Centrífugo </td><td> Beckman Coulter </td><td> Allegra X-12R </td><td></td></tr><tr><td> Espectrofotómetro </td><td> Varian </td><td> Cary 4000 UV-Visible </td><td></td></tr><tr><td> Láser </td><td> CNI Laser </td><td> MGL-532 </td><td></td></tr><tr><td> Micrómetro </td><td> Mitutoyo </td><td> Serie 227 </td><td></td></tr><tr><td> Microscopio quirúrgico </td><td> Zeiss </td><td> OPMI </td><td></td></tr></tbody></table>

fabrication and application of rose bengal-chitosan films in laser tissue repair

Adhesión tisular fotoquímica (PTB) es una técnica de sutura para la reparación de tejidos, que se consigue mediante la aplicación de una solución de rosa de bengala (RB) entre dos bordes de tejido 1,2. Estos son entonces irradiado por un láser que es absorbida selectivamente por la RB. Las reacciones fotoquímicas resultantes supuestamente reticular las fibras de colágeno en el tejido con una mínima producción de calor 3. En este informe, RB se ha incorporado en delgadas películas de quitosano para la fabricación de un adhesivo de tejido nuevo que es activado por láser. Las películas adhesivas, basado en quitosano y que contiene ~ 0,1% en peso de RB, se fabrican y se une a intestino de ternero y nervios tibiales de rata por un láser de estado sólido (λ = 532 nm, la fluencia ~ 110 J / cm 2, tamaño de la mancha ~ 0,5 cm) . Un tensiómetro sola columna, en interfaz con un ordenador personal, se utiliza para probar la resistencia de unión. El RB-quitosano adhesivas bonos firmemente al intestino con una resistencia de 15 ± 6 kPa, (n = 30). La fuerza de adhesión se reduce a 2 yplusmn; 2 kPa (n = 30) cuando el láser no se aplica al adhesivo. La anastomosis de los nervios tibial también se puede realizar sin el uso de suturas. Una novela quitosano adhesivo ha sido fabricado de que los bonos fotoquímicamente al tejido y no requiere suturas.

Watch this Scientific Journal Video about Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair at JoVE.com

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Las suturas son generalmente necesarios para reparar el tejido durante los procedimientos quirúrgicos. Sin embargo, su aplicación puede ser problemático, ya que son invasivos y pueden dañar el tejido. Los métodos de fabricación y aplicación de un adhesivo tisular novela se informó aquí. Esta película de adhesivo se activa por láser y no requiere el uso de suturas.

Fabricación y Aplicación de Rosa de Bengala-quitosano Films en Tissue Repair Laser

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

fabrication-application-rose-bengal-chitosan-films-laser-tissue

Research

JoVE Journal

Bioengineering

12.1K vistas.  University of Western Sydney, NSW Australia. Las suturas son generalmente necesarios para reparar el tejido durante los procedimientos quirúrgicos. Sin embargo, su aplicación puede ser problemático, ya que son invasivos y pueden dañar el tejido. Los métodos de fabricación y aplicación de un adhesivo tisular novela se informó aquí. Esta película de adhesivo se activa por láser y no requiere el uso de suturas.

Video: Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Fabricación de materiales de origen biológico inyectable para ingeniería de tejidos del miocardio

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Investigación

Bioingeniería

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Campo eléctrico controlado por migración dirigida de células progenitoras neurales en entornos 2D y 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Los tejidos de ingeniería músculo esquelético de células mioblastos murinos progenitoras y aplicación de estimulación eléctrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

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

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

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

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 ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adaptación del Proceso Electrohilado para proporcionar tres ambientes únicos para un Tri-capa<em&gt; In Vitro</em&gt; Modelo de la pared de la vía aérea

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tejido picado en colágeno comprimido: Un Bio Transplant que contiene células para la reparación reconstructiva-puesta en escena individual

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Guiada por imagen, fabricación basada en láser de Redes de microfluidos derivados-vascular

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabricación y caracterización de tejido óptico fantasmas que contiene macroestructura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imagen fotoacústica fotoacústica dual para visualización vascular

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...