Damos las gracias a Maegan B. Colehour para ayudar con el desarrollo bio-reactor. Estos estudios fueron financiados por la Universidad de Yale Departamento de Anestesia y por el NIH subvención HL 098 220 (a LEN). THP fue apoyado por el NIH T32 GM007171.

<ol>
	<li>American Lung Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lung Disease Data. , (2008).</li><li>Petersen, T. H., Calle, E. A., Colehour, M. B., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+for+the+Long+Term+Culture+of+Lung+Tissue.">Bioreactor for the Long Term Culture of Lung Tissue.</a> Cell Transplant. , (2010).</li><li>Bilek, A. M., Dee, K. C., Gaver, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+surface+tension+induced+epithelial+cell+damage+in+a+model+of+pulmonary+airway+reopening.">Mechanisms of surface tension induced epithelial cell damage in a model of pulmonary airway reopening.</a> J Appl Physiol. 94, 770-783 (2003).</li><li>Petersen, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-Engineerined+Lungs+for+in+Vivo+Implantation.">Tissue-Engineerined Lungs for in Vivo Implantation.</a> Science. 329, 538-5341 (2010).</li><li>Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat Med. 16, 927-9233 (2010).</li><li>Cortiella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+acellular+natural+lung+matrix+on+murine+embryonic+stem+cell+differentiation+and+tissue+formation.">Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.</a> Tissue Eng. Part A. 16, 2565-2580 (2010).</li><li>Sugihara, H., Toda, S., Miyabara, S., Fujiuyama, C., Yonemitsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+alveolus-like+structure+from+alveolar+type+II+epithelial+cells+in+three-dimensional+collagen+gel+matrix+culture.">Reconstruction of alveolus-like structure from alveolar type II epithelial cells in three-dimensional collagen gel matrix culture.</a> Am J Pathol. 142, 783-7892 (1993).</li><li>Choe, M. M., Sporn, P. H., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+remodeling+by+dynamic+strain+in+a+three-dimensional+tissue-engineered+human+airway+wall+model.">Extracellular matrix remodeling by dynamic strain in a three-dimensional tissue-engineered human airway wall model.</a> American Journal Respiratory Cell Molecular Biology. 35, 306-306 (2006).</li><li>Cortiella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-enginered+lung:+an+in+vivo+and+in+vitro+comparison+of+polyglycolic+acid+and+pluronic+F-127+hydrogel/somatic+lung+progenitor+cell+constructs+to+support+tissue+growth.">Tissue-enginered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth.</a> Tissue Engineering. 12, 1213-1213 (2006).</li><li>Price, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Decellularized+Lung+Bioreactor+System+for+Bioengineering+the+Lung:+The+Matrix+Reloaded.">Development of a Decellularized Lung Bioreactor System for Bioengineering the Lung: The Matrix Reloaded.</a> Tissue Eng Part A. , (2010).</li><li>Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. 14, 213-221 (2008).</li><li>Uygun, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat. Med. 16, 814-820 (2010).</li><li>Shannon, J. M., Mason, R. J., Jennings, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+of+alveolar+type+II+epithelial+cells+in+vitro:+effects+of+cell+shape,+cell-matrix+interactions+and+cell-cell+interactions.">Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions.</a> Biochim. Biophys. Acta. 931, 143-156 (1987).</li><li>Cortiella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Acellular+Natural+Lung+Matrix+on+Murine+Embryonic+Stem+Cell+Differentiation+and+Tissue+Formation.">Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation.</a> Tissue Eng Part A. , (2010).</li></ol>

LEN tiene acciones en Humacyte, una compañía de medicina regenerativa. Humacyte no financiar estos estudios, y no afectan al diseño, la interpretación, o la notificación de cualquiera de los experimentos o métodos descritos. Los autores (LEN, THP, EAC) y la Universidad de Yale ha presentado una solicitud de patente relacionada con la ingeniería de tejidos de los pulmones.

Los aspectos más críticos del sistema que aquí se presenta incluye el mantenimiento de la esterilidad, y la estrecha vigilancia de las presiones ejercidas sobre el lecho vascular en todo el proceso de preparación y siembra el andamio y el cultivo de la repoblación de pulmón. La esterilidad se mantiene mejor en autoclave todos los materiales antes de su uso, y mediante el montaje de los pulmones en un sistema cerrado poco después de explante y evitar el posterior incumplimiento de esta barrera. Después de la matriz descelularizada ha sido totalmente aclarado y se transfiere a un biorreactor estéril para la cultura, el tapón de silicona y otros sellos y las conexiones no debe ser alterado o eliminado. Para mantener la presión bajo control, el flujo por gravedad, es preferible siempre que sea posible. Cuando una bomba es necesaria para la recirculación del fluido, que comienzan después del lavado con PBS y continua durante el cultivo, se recomienda medir directamente la presión justo antes de que el líquido entra en la arteria pulmonar con un transductor de presión. La magnitud de la presión aplicada no debe exceder los 15 mm Hg. Recellularized pulmones pueden ser cultivados durante períodos variables de tiempo, por lo general van de 4 días a un máximo de 3 semanas. Perfusión vascular se realiza generalmente en 1-3 ml / min durante el cultivo del endotelio, mientras que la ventilación se aplica típicamente a una velocidad de 1 respiración / min durante el cultivo epiteliales. Durante los periodos de la cultura combinados, la ventilación y la perfusión simultánea es la adecuada. La ventilación se puede realizar con cualquier medio líquido o aire. En el transcurso de las últimas décadas, varios grupos han realizado una importante labor de ingeniería de tejidos que muestran la viabilidad de la diferenciación del epitelio pulmonar in vitro y de reproducir varios aspectos de pulmón microanatomía 7,8,9, 10. Sin embargo, hasta hace poco, ninguno de estos intentos de 4,5 para diseñar el tejido pulmonar se había traducido en un órgano implantable que fue capaz de mantener la separación entre la sangre y los compartimentos de las vías respiratorias y que pueden participar en el intercambio de gases. Por lo tanto, aunque los métodos descritos son sólo un primer paso hacia el objetivo a largo plazo de la generación de tejido pulmonar funcional, este trabajo es un paso alentador hacia la posibilidad de aumentar la cantidad de tejido pulmonar disponible para el trasplante. Por otra parte, este trabajo elabora el trabajo realizado por Ott et al. Uygun y sus colegas y 11,12, lo que demuestra la eficacia de una matriz extracelular descelularizada como un andamio para la ingeniería de tejidos tridimensionales complejas estructuras y apoyar el crecimiento y la supervivencia de varios tipos de células . Este trabajo también es importante por su contribución al arsenal terapéutico de las células respiratorias y biólogos moleculares. Al ofrecer un único entorno tridimensional que también puede proporcionar adecuados estímulos mecánicos, y que no comparte el riesgo concomitante de una rápida des-diferenciación que se puede encontrar al epitelio de cultivo de los roedores en el laboratorio con los métodos más tradicionales 13, los científicos podrían usar nuestro sistema para obtener nuevos conocimientos sobre las interacciones célula-célula y célula-matriz que juegan un papel en la diferenciación y función celular. Este conocimiento puede ser muy poderosa si se usa como palanca para guiar el destino de varias poblaciones de células madre, como grupo Cortiella ha demostrado en los estudios iniciales 14.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Eutanasia </td><td></td><td></td><td></td></tr><tr><td> Heparina </td><td> Sigma-Aldrich </td><td> H4784 </td><td></td></tr><tr><td> Nitroprusiato de sodio </td><td> Fluka </td><td> 71778 </td><td></td></tr><tr><td> Euthasol solución de la eutanasia </td><td> Virbac AH </td><td> 710101 </td><td> 390 mg / ml de solución de pentobarbital de sodio de valores debe ser diluido apropiadamente para la administración de IP </td></tr><tr><td colspan="4"></td></tr><tr><td> Decell Solución </td><td></td><td></td><td></td></tr><tr><td> CHAPS </td><td> Sigma-Aldrich </td><td> C3023 </td><td></td></tr><tr><td> NaCl </td><td> Estadounidense Bioanalytical </td><td> AB01915 </td><td></td></tr><tr><td> EDTA </td><td> Sigma </td><td> E5134 </td><td></td></tr><tr><td> NaOH </td><td> JT Baker </td><td> 3722-01 </td><td></td></tr><tr><td> 1X PBS </td><td> Gibco </td><td> 14190 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td> Componentes biorreactor </td><td></td><td></td><td></td></tr><tr><td> 480 ml frasco </td><td> Cole Parmer </td><td> EW-3460560 </td><td></td></tr><tr><td> Tapón de silicona, talla 14 </td><td> Cole Parmer </td><td> EW-06.298-26 </td><td></td></tr><tr><td> Y-conectores </td><td> Cole Parmer </td><td> ED-30.614-08 </td><td> Se utiliza para cánulas arteriales y traqueal </td></tr><tr><td> Tubo de silicona </td><td> Masterflex </td><td> 96420-14, 16 </td><td> L / S 14, 16 </td></tr><tr><td> Transductores de presión </td><td> Edwards Lifesciences </td><td> PX-212 </td><td></td></tr><tr><td> La válvula de retención </td><td> Cole Parmer </td><td> EW-98.553-20 </td><td> Válvula de una vía </td></tr><tr><td> Llaves de paso de 4 vías </td><td> Edwards Lifesciences </td><td> 594WSC </td><td></td></tr><tr><td> Filtro de jeringa </td><td> Cole Parmer </td><td> 2915-08 </td><td> PTFE, 0.2μm </td></tr><tr><td colspan="4"></td></tr><tr><td> Decell y apparati enjuague (adiciones al biorreactor) </td><td></td><td></td><td></td></tr><tr><td> 500 y 1000 ml botellas de vidrio </td><td> Corning </td><td> 1395-500;-1L </td><td> Se utiliza para decell y aclarado por la gravedad </td></tr><tr><td colspan="4"></td></tr><tr><td> Benzonase tratamiento </td><td></td><td></td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> Gibco </td><td> 15140122 </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td> Tris-HCl </td><td> Estadounidense Bioanalytical </td><td> AB14043 </td><td> 1 M, pH 8,0 </td></tr><tr><td> MgCl2 </td><td> JT Baker </td><td> 2444-01 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A9647 </td><td></td></tr><tr><td> Benzonase nucleasa </td><td> Sigma </td><td> E1014 </td><td> Endonucleasa utiliza para eliminar restos de ADN de la matriz </td></tr></tbody></table>

procedure for lung engineering

Los tejidos pulmonares, incluyendo cáncer de pulmón y enfermedades pulmonares crónicas, como enfermedad pulmonar obstructiva crónica, de forma acumulativa representan alrededor de 280.000 muertes al año, la enfermedad pulmonar obstructiva crónica es actualmente la cuarta causa de muerte en los Estados Unidos 1. Que contribuyen a esta mortalidad es el hecho de que los pulmones por lo general no más allá de reparar o regenerar el nivel microscópico, celular. Por lo tanto, el tejido pulmonar que está dañado por la degeneración o una infección, o el tejido pulmonar resecado quirúrgicamente que no es funcionalmente reemplazado en vivo. Para explorar si el tejido pulmonar se pueden generar in vitro, se trataron los pulmones de ratas adultas utilizando un procedimiento que elimina los componentes celulares para producir un andamio extracelular pulmonar acelular matriz. Esta estructura mantiene las estructuras jerárquicas de ramificación de las vías respiratorias y los vasos sanguíneos, así como una membrana basal en gran parte intacto, que comprende colágeno IV, laminina y fibronectina. El andamio se monta en un biorreactor diseñado para imitar los aspectos críticos de la fisiología pulmonar, tales como ventilación con presión negativa y la perfusión vascular pulsátil. Por el epitelio pulmonar y el cultivo de endotelio vascular en el andamio montado en biorreactores, que son capaces de generar tejido pulmonar que es fenotípicamente similar al tejido pulmonar de origen y que es capaz de participar en el intercambio de gases para los intervalos de tiempo (45-120 minutos). Estos resultados son alentadores y sugieren que la repoblación de la matriz pulmonar es una estrategia viable para la regeneración del pulmón. Esta posibilidad se presenta una oportunidad no sólo de trabajar para aumentar la oferta de tejidos de los pulmones para un trasplante, sino también para el estudio de células respiratorias y la biología molecular in vitro durante más tiempo y en un microambiente más preciso que previamente ha sido posible.

Watch this Scientific Journal Video about Procedure for Lung Engineering at JoVE.com

Procedure for Lung Engineering

Hemos desarrollado una matriz extracelular y de pulmón descelularizada biorreactor novela biomiméticos que se puede utilizar para generar tejido pulmonar funcional. Por la siembra de las células en la matriz y el cultivo en el biorreactor, que generan tejido que demuestra el intercambio de gases eficaz cuando se trasplantan en vivo por periodos cortos de tiempo.

Procedimiento para la Ingeniería de pulmón

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

46.6K vistas.  Yale University. Hemos desarrollado una matriz extracelular y de pulmón descelularizada biorreactor novela biomiméticos que se puede utilizar para generar tejido pulmonar funcional. Por la siembra de las células en la matriz y el cultivo en el biorreactor, que generan tejido que demuestra el intercambio de gases eficaz cuando se trasplantan en vivo por periodos cortos de tiempo.

Video: Procedure for Lung Engineering

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

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomérico andamios en ingeniería de tejidos arteriales

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproducción de procesamiento de las Fibras electrospun de Ingeniería de Tejidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Fuerza Litografía Cardiaca de Ingeniería de Tejidos

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Síntesis de nanofibras a base de Queratina de Ingeniería Biomédica

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparación de Superficies de respuesta térmica nanoestructurados para la Ingeniería de Tejidos

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularizado de Bioprinting de microfluidos para la ingeniería de tejidos y organitas

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Un protocolo para Decellularizing ratón cócleas para ingeniería de tejidos del oído interno

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Dos métodos de descelularización de tejidos vegetales para aplicaciones de ingeniería de tejidos

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...