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 border="0" cellpadding="0" cellspacing="0">
	<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>&#945;-Modified Eagle&#39;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

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

De alta resolução de fibra óptica para Microendoscopy In situ Celular imagem

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Bioengenharia

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 Andaimes em Engenharia de Tecidos Arterial

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.

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 ...

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 ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Repovoamento celular epitelial e Preparação de Rodent Matriz Extracelular Andaimes para o desenvolvimento do tecido renal

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

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.

Síntese de nanofibras baseado em queratina de Engenharia Biomédica

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

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

A Facile e Eco-friendly rota para fabricar poli (ácido láctico) Scaffolds com Graded Pore Tamanho

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Derreter a eletrofiação escrita de andaimes Poly(ε-caprolactone) tridimensional com morfologias controláveis para aplicações de engenharia de tecidos

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Fabricação e caracterização de andaimes de fibra Griffithsin-modificado para a prevenção de doenças sexualmente transmissíveis

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Cultivo de células de mamíferos em andaimes tridimensionais do Peptide

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...