Este trabalho foi financiado parcialmente com fundos de arranque da Universidade de Nebraska Medical Center e National Institute of Health (número de concessão 1R15 AR063901-01).

1. Preparação da Solução <ol><li> Prepara-se uma solução de poli (ε-caprolactona) (PCL) (M w = 80.000 g / mol) a uma concentração aproximada de 100 mg / ml. Dissolver PCL numa mistura de diclorometano (DCM) e N, N-dimethlyformamide (DMF), a uma proporção de 4: 1 (v / v) com uma concentração de 10% (w / v). </li><li> Colocar a solução em um tubo de vidro de 20 ml para a mistura. Coloque tubo de vidro em limpador ultra-sônico por 30 minutos, ou até que a solução é translúcida. </li></ol> 2. Preparação Aparelho <ol><li> Adicionar a solução PCL preparado em uma seringa de 5 ml com uma agulha romba de calibre 21 em anexo. </li><li> Coloque bomba de seringa numa posição vertical, electrospinning, de acordo com a Figura 1. </li><li> Use um coletor de aço inoxidável-gap com um espaço aberto de 2 cm x 5 cm para o substrato de fibra alinhada. Colocar o colector 12 cm depois da ponta da agulha. </li><li> Ligue a corrente contínua (DC) de alta tensão da fonte de alimentação para oagulha e aterrar o colecionador. Certifique-se de que o coletor é aterrado individualmente, sem contato com o outro equipamento de laboratório. </li></ol> 3. Electrospinning <ol><li> Conjunto de bomba de seringa a 1,50 ml / hr, até que as gotículas que formam a ponta da agulha são substituídas imediatamente, removido. Em seguida, defina a taxa de fluxo de 0,50 ml / hr. </li><li> Vire o operador de tensão a 12 kV. </li><li> Electrospin até que as fibras uniaxialmente alinhados cobrir completamente a lacuna no coletor. </li><li> Aplicar cola nas bordas de uma pequena placa de vidro e transferir as fibras a partir do colector abertura para a placa de vidro. Colocar a placa de vidro na parte superior de um pedaço de folha de alumínio que tem o mesmo tamanho que a placa de vidro e é ligada à terra. </li><li> Posicionar o segundo colector de seringa de acordo com a Figura 3. </li><li> Fixe a máscara de plástico para a segunda bomba de seringa e posicioná-lo de 2 mm acima do coletor. </li><li> Adicionar Cumarina 6 a 1% (w / w) para a solução de PCL-se nanoencapsulation é desejado-e misturar até a solução estar translúcida. </li><li> Coloque o PCL ou PCL / Coumarin 6 solução em uma agulha 5 ml com um blunt agulha de calibre 21. Definir bomba vertical para 0,50 ml / hr e horizontal para puxar a 9 ml / h ou a uma velocidade aproximada de 1 mm / min. </li><li> Electrospin até que a máscara se mudou quase completamente para fora do colector, mas com a área da borda ainda sob a máscara. </li></ol> 4. Fiber Caracterização <ol><li> Colocar as amostras de fita condutora dupla face ao pino metálico e revestimento com platina por 40 seg usando um revestidor por crepitação a 40 mA. <ol><li> Examine fibras através do microscópio eletrônico de varredura (SEM) de acordo com os nossos estudos anteriores 17,18. </li><li> Coletar imagens a uma tensão de aceleração de 15 kV. </li></ol></li><li> Realizar análise rápida de Fourier (FFT) em uma amostra de fibra separada para medir o alinhamento de fibras. As informações detalhadas sobre o alinhamento das fibras de medição por FFT pode referir-se to estudos anteriores 19,20. </li></ol> 5. As células-tronco Sementeira. <ol><li> Esteriliza-se as amostras de fibras por imersão em uma solução de etanol a 70% durante 2 horas. Em seguida, lavar as fibras com água destilada para remover quaisquer contaminantes. </li><li> Obter ADSCs humanos e em células de cultura de um frasco de 25 cm2 a 37 ° C numa atmosfera de 95% ar / 5% de CO 2. Mudar o meio de cultura de células em dias alternados 10. </li><li> Tripsinizar as células e contagem do número de células. Especificamente, remover todo o meio de cultura a partir do balão de cultura de células e lava-se as células com solução salina tamponada com fosfato (PBS) duas vezes. Em seguida, adicionar 1 m l de uma solução a 0,25% de tripsina-EDTA (pré-aquecido no banho de água a 37 ° C) para cobrir a monocamada de células e incuba-se o balão no incubador de cultura de células durante 2-3 minutos. Adicionar 4 ml de meio de cultura e lava-se a todas as células a partir da superfície por meio da pipetagem de toda a superfície. Th Centrifugar a suspensão de células e re-dispersae o sedimento de células em meio de cultura. Contagem de células, via hemacitómetro. Semente de cerca de 1 x 10 4 células para o andaime colocado em nanofibras de 35 mm -cultura prato e incubar durante 3 a 7 dias. </li><li> Células mancha utilizando diacetato de fluoresceína (5 mg / mL em acetona) (FDA), em seguida, as amostras de imagem usando um microscópio de fluorescência. Especificamente, adicionar 100 ul de solução FDA para o prato de cultura e incuba-se durante 30 min. Lavar as células com PBS três vezes antes de imagem fluorescente. Analisar orientação celular por um programa de mat-lab personalizadas com base nos nossos estudos anteriores 10. </li></ol>

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The authors have nothing to disclose.

The most critical part of the protocol is generation of the gradient scaffold. It is imperative that the mask covering the collector moves at a constant velocity so there is a gradual change within the fiber scaffold. The correct preparation of PCL solution is also important to ensure electrospinning success. Checking the fiber morphology prior to electrospinning is recommendable, especially after the encapsulation of Coumarin-6, which may require a higher voltage to electrospin correctly. 
Fu...

Nanofibras são um utilitário popular para engenharia de tecidos, devido à sua capacidade de imitar a matriz extracelular na sua estrutura e tamanho relativo 1. No entanto, algumas interfaces de tecido nativo, tais como o local de inserção do tendão para osso, contêm fibras de colagénio, os quais exibem uma estrutura organizacional variável que aumenta na direcção de alinhamento do tendão e diminui no local do osso 2-5. Assim, para a regeneração de tecidos eficaz, há uma necessidade de fabricar uma estrutura de suporte que pode efectivamente mimetizar este gradiente estrutural. Anteriormente, houve uma pesquisa conduzida em mudanças graduais na composição de fibra, mais especificamente, o teor mineral 6. No entanto, a recriação da componente estrutural dos tecidos conjuntivos permanece largamente inexplorado. Um estudo anterior examinada gradientes morfológicas estudando o efeito da densidade de partículas de sílica de superfície sobre a proliferação de osteoblastos de calvárias de rato e encontrou um inverse relação entre a densidade de partículas de sílica e a proliferação de células 7. Mas as mudanças morfológicas que mediaram a proliferação celular em trabalhos anteriores eram em sua maioria relacionada à rugosidade superficial falta a capacidade de imitar fibra de mudanças organizacionais 7,8. Um estudo recente tentativa de fabricar um andaime que imitava as orientações de fibras colágenas originais usando um romance coletor para eletrofiação 9. Embora este estudo conseguiu produzir um andaime com fibras de ambos alinhados e aleatórios, não conseguiu imitar as mudanças graduais exibidas nos tecidos nativos. Além disso, na produção de componentes separados, com uma mudança imediata da alinhado com orientação aleatória, as propriedades biomecânicas do presente andaime diminuiu significativamente. Nenhum trabalho anterior foi capaz de produzir scaffolds nanofibras aplicáveis ​​com gradações contínuas em orientações de fibra de alinhados e aleatória. Nosso estudo recente mostrou recreação sucesso de andaimes de nanofibrascom gradações de organização das fibras que podem, potencialmente, imitam a organização do colágeno nativo no tendão-de-osso de inserção 10. Este trabalho tem como objetivo apresentar os protocolos utilizados para a produção de andaimes de nanofibras com uma estrutura que se assemelha a de organização das fibras na interface do tecido do tendão-de-osso nativo. Estruturas de nanofibras Gradiente têm potencialmente de longo alcance aplicações em uma variedade de campos. Estamos focados em aplicações para engenharia de tecidos do local de inserção do tendão-de-osso através da combinação de nossos andaimes com células-tronco derivadas de tecido adiposo (ADSCs) que já são utilizadas para a regeneração de tecidos em vários substratos 11-14. Além disso, ADSCs são muito semelhantes na natureza para as células-tronco da medula óssea em termos de multipotency e os recursos são abundantes que podem ser colhidas usando um simples 15,16 procedimento de lipoaspiração. Semeando destas células para andaimes nanofibras gradativas aumenta ainda mais a sua tisprocessar as aplicações de engenharia, permitindo a distribuição controlada das células que podem, potencialmente, diferenciam-se em vários tecidos. Além disso a sementeira de células estaminais, nanofibras pode ser encapsulado com moléculas de sinalização para a regulação da resposta celular. Acoplamento nanoencapsulação com o gradiente de organização destes suportes permite o estudo do comportamento celular ou possíveis modelos de implantes e os revestimentos. A encapsulação de moléculas funcionais como proteína morfogenética óssea 2 (BMP-2), que tem sido mostrado induzir a diferenciação dos osteoblastos 15,16, poderia aumentar ainda mais as aplicações de engenharia de tecidos destes suportes 10.

<table><tbody><tr><td>Polycaprolactone</td><td>Sigma-Aldrich</td><td>440744</td><td></td></tr><tr><td>N,N-Dimethlyformamide</td><td>Fisher Chemical</td><td>D-119-1</td><td></td></tr><tr><td>Dichloromethane</td><td>Fisher Chemical</td><td>AC61093-1000</td><td></td></tr><tr><td>Coumarin 6</td><td>Sigma-Aldrich</td><td>546283</td><td></td></tr><tr><td>Adipose Derived Stem Cells</td><td>Cellular engineering Technologies</td><td>HMSC.AD-100</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>Fluorescein Diacetate</td><td>Sigma-Aldrich</td><td>F7378</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td></td></tr><tr><td>&amp;alpha;-Modified Eagle&#039;s Medium</td><td>Invitrogen</td><td>a10490-01</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>s25120a</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Invitrogen</td><td>10010023</td><td></td></tr><tr><td>Glass Slides</td><td>VWR international, LLC</td><td>101412-842</td><td></td></tr><tr><td>Syringe Pump</td><td>Fisher Scientific</td><td>14-831-200</td><td>Single syringe</td></tr><tr><td>Ultrasonic Cleaner</td><td>Branson</td><td>1510</td><td></td></tr><tr><td>High Voltage DC Power Supply</td><td>Gamma High Voltage Research</td><td>ES30</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>FEI</td><td>Nova 2300</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Zeiss</td><td>Axio Imager 2</td><td></td></tr></tbody></table>

electrospun nanofiber scaffolds with gradations in fiber organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Usando este protocolo, um tapete de fibra com um gradiente de organização foi formada. A Figura 3 mostra as imagens SEM tomadas em vários locais no cadafalso nanofibras. Qualitativamente, pode ser determinado que não existe uma progressão das fibras alinhadas uniaxialmente a 0 mm (Figura 3A) para uma variedade de fibra casual aos 6 mm (Figura 3D). A FFT dá um valor quantitativo para o alinhamento de fibras, detalhes sobre os processos quantitativos são detalhados...

Watch this Scientific Journal Video about Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization at JoVE.com

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Aqui, apresentamos um protocolo para fabricar andaimes electrospun nanofibras com organização gradated de fibras e explorar suas aplicações na regulação da morfologia celular / orientação. Gradientes no que diz respeito às propriedades físicas e químicas dos scaffolds nanofibras oferecer uma ampla variedade de aplicações na área biomédica.

Electrospun nanofibras Andaimes com gradações de Organização Fiber

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

electrospun-nanofiber-scaffolds-with-gradations-in-fiber-organization

Research

JoVE Journal

Bioengineering

9.8K visualizações.  University of Nebraska Medical Center.  Aqui, apresentamos um protocolo para fabricar andaimes electrospun nanofibras com organização gradated de fibras e explorar suas aplicações na regulação da morfologia celular / orientação. Gradientes no que diz respeito às propriedades físicas e químicas dos scaffolds nanofibras oferecer uma ampla variedade de aplicações na área biomédica. 

Vídeo: Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Fundamentos eletrofiação: Solução de Otimização e Parâmetros Aparelho

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

Bioengenharia

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

A Cultura da Motor Primário e neurônios sensoriais no Media Definidas em electrospun Poly-L-ácido lático nanofibras Andaimes

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Neurociência

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.

Processamento de pós-produção de Fibras electrospun para Engenharia de Tecidos

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

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Expansão de duas dimensões Electrospun nanofibras esteiras em três dimensões andaimes

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

Genética

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.

Andaimes auto-relato para cultura de células 3-Dimensional

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

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

Nanofibras ECM proteínas e Nanoestruturas Engineered Usando Assembléia iniciou-Surface

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Electrospinning Fator de Crescimento Releasing Microesferas em fibrosa Andaimes

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Electrospun fibrosa Andaimes de Poli (glicerol-dodecanedioate) para Engenharia Neural tecidos de rato as células estaminais embrionárias

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

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.

Adaptação do Processo Electrospinning para fornecer três ambientes únicos para um Tri-camadas<em&gt; In Vitro</em&gt; Modelo da parede das vias aéreas

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

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

Eletrofiação Andaimes Polymer fibrosos para Engenharia de Tecidos e Cultura de Células

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...