Este trabalho foi financiado pelo NIH K08 EB003996 e os Veteranos Paralisados ​​da América Research Foundation Grant 2573.

1. Escolha um Polímero <ol><li> Escolha um polímero (por exemplo, poli-L-láctico (PLLA), policaprolactona (PCL), poliestireno (PS) ou nylon) com base em suas especificações (por exemplo, biodegradável, termoplástico ou cross-linkable) e um solvente que de polímero. Escolha o equipamento de protecção individual adequado com base em sua seleção. </li><li> Selecione um substrato com base na sua aplicação (por exemplo, pastilha de vidro, metal, plástico ou silicone). </li></ol> 2. Escolha um coletor <ol><li> Escolha a geometria do coletor com base em suas especificações. Fibras aleatórias podem ser coletados em placas estacionárias. Fibras alinhadas podem ser coletados em rotação rápida rodas, tambores ou varas, ou em placas paralelas. </li><li> O coletor deve ser condutora e deve permanecer isolada do seu eixo de tal forma que ele pode ser aterrado sem aterramento também objetos adjacentes, o tampo da mesa, etc </li></ol> 3. Aproximar as Concentração Entanglement Critical Empiricamente 1 <ol><li> Prepare concentrações candidato várias polímero (por exemplo, 4, 10, 15, 20, 30% em peso) e escolha uma concentração que flui (a solução deve ser um líquido viscoso, mas não um gel) para começar. </li><li> Configurar o aparelho electrospinning 2,3,4,5 (ver Figura 1) <ol><li> Carregar a bomba de seringa e definir a velocidade da bomba de tal forma que qualquer gota de solução limpou a partir da ponta é imediatamente substituído. </li><li> Coletor o solo eo grampo do fio de alta tensão para a placa de condutores (um pequeno quadrado de material condutor, como folha de alumínio através da qual se projeta a ponta da seringa). </li><li> Começar a girar a roda. </li><li> Certifique-se o fornecimento de energia está definido para zero antes de ligá-lo. </li></ol></li><li> Observar o fluxo <ol><li> Rampa de tensão se devagar e ver o talão da solução na ponta da agulha. </li><li> Ajustar a tensão para obter um fluxo duradouro e estável. Se um fluxo constante não pode ser obtida, ajustar a concentração da solução de polímero. Ver Tabela 1 para um exemplo. </li></ol></li></ol> 4. Solução de problemas - o Stream: <ol><li> Problemas para visualizar o fluxo de <ol><li> Use um pano de fundo fosco escuro e coloque uma fonte de luz unidirecional (como uma lanterna) entre o espectador eo fluxo (ver figura 2). </li></ol></li><li> Pingando da ponta da seringa <ol><li> Se a solução de polímero está gotejando para baixo com nenhuma atração para a roda, certifique-se o prato condutor está fazendo contato com a ponta da agulha e que o coletor está fazendo contato com o solo. </li><li> Se a gota de polímero solução na ponta da seringa está inclinado na direção da roda, mas não está formando uma corrente, aumentar a tensão. A qualidade do fluxo pode ser ajustado pela variação da distância e da tensão até um fluxo constante é visível. Veja a Figura 3 para distâncias sugeridas com tensões correspondentes para uma solução de PLLA 4% e uma placa de condutores 8x8 cm. </li></ol></li><li> Globs grande na ponta da seringa <ol><li> Quando a solução de polímero começa a coletar e endurecer na ponta da agulha de furto, o glob afastado com uma toalha de papel anexado a uma vara não-condutor. </li></ol></li><li> Oscilante ou &#39;abanar&#39; streams Quando o fluxo está abanando cima e para baixo rapidamente, diminua a tensão ou aumentar a distância entre a ponta da seringa e da roda. Se o fluxo continua a abanar utilizar uma maior concentração de polímero ou adicionar um pouco de solvente a uma menor evaporação. </li><li> Fluxos de curto ou descontínuo Visível córregos constantes fazer contato observáveis ​​com o conjunto roda em alta velocidade de rotação de rendimento a mais alta qualidade de uniformidade e alinhamento. Quando o fluxo é curta e descontínua, aumentando a solução de polímero, adicionando mais lenta evaporação do solvente, e ajustar a tensão vai melhorar o comprimento ea estabilidade do fluxo. </li></ol> 5. Solução de problemas - Morfologia Fiber 6,7,8 (veja a Figura 4) <ol><li> Beading Quando contas são descobertos em fibras, aumentar a solução de polímero e certifique-se o prato condutor está fazendo contato contínuo com a agulha e que a escova de fio terra está fazendo contato contínuo com a roda. </li><li> Fitas e fibras de sangramento Quando as fibras estão se formando como fitas ou estão sangrando juntas, use uma maior concentração de polímero ou um solvente com uma maior taxa de evaporação (mais voláteis). </li><li> Fibras Curlicue ou ondulado Quando as fibras estão formando ondas ou arabescos, aumentar a velocidade da roda ou mover a ponta da agulha mais do coletor. Além disso, verifique se a placa de condutores e coletor não estão vibrando. </li><li> Porosidade 9 Se os poros são desejados, use um solvente evapora rapidamente. Se os poros não são desejados, tente adicionar uma pequena quantidade de co-solvente, que é menos volátil do que o solvente principais. </li><li> Alinhamento 10 Quando o coletor é moving em baixas rotações ou em repouso, a qualidade de alinhamento é pobre. Aumentar o alinhamento, aumentando a velocidade da roda. </li></ol> 6. Resultados representativos: Por favor, veja a Figura 4 para representações de resultados de fibra típica. <img src="/files/ftp_upload/2494/2494fig1.jpg" alt="Figura 1" /> Figura 1. A configuração electrospinning típico. A solução de polímero (azul) está dispensada de uma bomba de seringa (laranja). A alta tensão de alimentação DC (verde) motivos um coletor de rodas de rotação rápida (cinza), sobre o qual nanofibras alinhadas são coletados. O jato de polímero entre a seringa eo coletor consiste de um segmento de constante fluxo e um segmento rapidamente oscilante chicotadas. <img src="/files/ftp_upload/2494/2494fig2.jpg" alt="Figura 2" /> . Figura 2 O jet streaming é visível sair da ponta da seringa; o jato chicotadas é muito pequeno para ser visto. Aproximar a concentração entrelaçamento crítico de PLLA <table border="1"><tbody><tr><td> PLLA (% wt / v) </td><td> Observação </td><td> Ajuste de concentração </td></tr><tr><td> 0,5 </td><td> Gotejamento, sem fluxo </td><td> Aumento </td></tr><tr><td> 2,0 </td><td> Cuspindo globs pequena, sem fluxo </td><td> Increse ligeiramente </td></tr><tr><td> 4,0 </td><td> Fluxo constante </td><td> Bom </td></tr><tr><td> 6 </td><td> Cuspindo globs grandes ou contas </td><td> Diminuir ligeiramente </td></tr><tr><td> 12,0 </td><td> Aglomeração na ponta; nenhum fluxo </td><td> Diminuir </td></tr></tbody></table> Tabela 1. Um exemplo que descreve a aproximação da concentração entrelaçamento crítico de PLLA. Várias concentrações de polímero são julgados e os jatos resultantes de streaming observado até um fluxo constante é obtida. <img src="/files/ftp_upload/2494/2494fig3.jpg" alt="Figura 3" /> Figura 3. A distância entre a ponta da seringa eo coletor deve ser equilibrada com a tensão aplicada para obter um jato constante streaming. Tensão aplicada em excesso provoca uma oscilação ou jet &#39;abanar&#39; a forma que resulta em menos fibras bem alinhadas. Quando a tensão é muito baixa, não vai jet forma ea solução só por gotejamento a partir da ponta da seringa. A região sombreada roxa acima representa a faixa de tensão sobre a qual um jato constante de streaming pode ser obtido para PLLA em função da seringa para coletor de distância. <img src="/files/ftp_upload/2494/2494fig4.jpg" alt="Figura 4" /> Fibras Figura 4. Electrospun podem apresentar uma variedade de morfologias, incluindo beading (A), fitas (B), (C) arabescos, globs porosos (D), bom alinhamento (E) e mau alinhamento (F).

<ol>
	<li>Shenoy, S. L., Bates, W. D., Frisch, H. L., Wnek, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+chain+entanglements+on+fiber+formation+during+electrospinning+of+polymer+solutions:+good+solvent,+non-specific+polymer-polymer+interaction+limit.">Role of chain entanglements on fiber formation during electrospinning of polymer solutions: good solvent, non-specific polymer-polymer interaction limit.</a> Polymer. 46, 3372-3384 (2005).</li><li>Gertz, C. C., Leach, M. K., Birrel, L. K., Martin, D. C., Feldman, E. L., Corey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+neuritogenesis+and+maturation+of+primary+spinal+motor+neurons+in+response+to+nanofibers.">Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers.</a> Dev. Neurobiol. 70, 589-603 (2010).</li><li>Lin, D. Y., Johnson, M. A., Vohden, R. A., Chen, D., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+nanofiber+morphologies+using+modulated+electrospinning+for+biomedical+applications.">Tailored nanofiber morphologies using modulated electrospinning for biomedical applications.</a> Mat. Res. Soc. Symp. Proc. 736, D3.8.1-D3.8.6 (2003).</li><li>Corey, J. M., Gertz, C. C., Wang, B. S., Birrell, L. K., Johnson, S. L., Martin, D. C., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+electrospun+PLLA+nanofiber+scaffolds+compatible+with+serum-free+growth+of+primary+motor+and+sensory+neurons.">The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons.</a> Acta. Biomater. 4, 863-875 (2008).</li><li>Corey, J. M., Lin, D. Y., Mycek, K. B., Chen, Q., Samuel, S., Feldman, E. L., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+electrospun+nanofibers+specify+the+direction+of+dorsal+root+ganglia+neurite+growth.">Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth.</a> J. Biomed. Mater. Res. A. 83, 636-645 (2007).</li><li>Tan, S. -. H., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+parameter+study+for+ultra-fine+fiber+fabrication+via+electrospinning+process.">Systematic parameter study for ultra-fine fiber fabrication via electrospinning process.</a> Polymer. 46, 6128-6134 (2005).</li><li>Yang, F., Murugan, R., Wang, S., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+nano/micro+scale+poly(L-lactic+acid)+aligned+fibers+and+their+potential+in+neural+tissue+engineering.">Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering.</a> Biomaterials. 26, 2603-2610 (2005).</li><li>Li, W., Laurencin, C. T., Caterson, E. J., Tuan, R. S., Ko, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+structure:+A+novel+scaffold+for+tissue+engineering.">Electrospun nanofibrous structure: A novel scaffold for tissue engineering.</a> J. Biomed. Mater. Res. A. 60, 613-621 (2002).</li><li>Kim, C. H., Jung, Y. H., Kim, H. Y., Lee, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+collector+temperature+on+the+porous+structure+of+electrospun+fibers.">Effect of collector temperature on the porous structure of electrospun fibers.</a> Macromol. Res. 14, 59-65 (2006).</li><li>Wang, H. B., Mullins, M. E., Cregg, J. M., Hurtado, A., Oudega, M., Trombley, M. T., Gilbert, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+highly+aligned+electrospun+poly-L-lactic+acid+fibers+for+nerve+regeneration+applications.">Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications.</a> J. Neural Eng. 6, (2009).</li></ol>

Não há conflitos de interesse declarados.

Nota: A maioria dos exemplos aqui apresentados lidar com electrospinning poli-L-láctico (PLLA) nanofibras. Isto é simplesmente porque é o polímero PLLA mais comumente girada em nosso laboratório. No entanto, também temos utilizado com sucesso destes métodos para electrospin outros polímeros (por exemplo, PLGA, PCL, PS) e acreditam que as técnicas aqui apresentadas são facilmente aplicáveis ​​para a maioria de médio a alto peso molecular de soluções de polímeros.

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>High voltage DC power supply</td>
<td>&nbsp;</td>
<td>Gamma High Voltage</td>
<td>ES40P-5W</td>
<td>&nbsp;</td>
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<td>Syringe pump</td>
<td>&nbsp;</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>&nbsp;</td>
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<td>Aluminum foil</td>
<td>&nbsp;</td>
<td>Reynolds</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Blunt metal tips, 23ga</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-850-102 </td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309585</td>
<td>&nbsp;</td>
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<td>Rotating or stationary collector</td>
<td>&nbsp;</td>
<td>Custom built</td>
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<td>Various alligator clips and wires</td>
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<td>Dimethylformamide</td>
<td>&nbsp;</td>
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<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC42355-0040 </td>
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<td>PLLA</td>
<td>&nbsp;</td>
<td>Boehringer Ingelheim</td>
<td>Resomer L210</td>
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<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
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<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13073-1</td>
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electrospinning fundamentals: optimizing solution and apparatus parameters

Andaimes electrospun nanofibras foram mostrados para acelerar a maturação, melhorar o crescimento, e direcionar a migração de células in vitro. Eletrofiação é um processo em que um polímero é cobrado jet coletados em um coletor aterrado; uma rápida rotação resultados coletor em nanofibras alinhadas enquanto resultado coletores estacionários em esteiras de fibra orientados aleatoriamente. O jato de polímero é formado quando uma carga eletrostática aplicada supera a tensão superficial da solução. Há uma concentração mínima para um dado polímero, chamado de concentração de emaranhamento crítico, abaixo do qual um jato estável não pode ser alcançado e não nanofibras formará - embora as nanopartículas podem ser alcançados (electrospray). Um jato estável tem dois domínios, um segmento de streaming e um segmento de chicotadas. Enquanto o jato chicotadas é geralmente invisíveis a olho nu, o segmento de transmissão é muitas vezes visível sob condições de iluminação apropriada. Observando-se o comprimento, espessura, consistência e movimento do fluxo é útil para prever o alinhamento e morfologia do nanofibras sendo formado. Um fluxo de curto, não uniforme, inconsistente e / ou oscilante é indicativo de uma variedade de problemas, incluindo o alinhamento de fibra pobres, beading, splattering e padrões curlicue ou ondulado. O fluxo pode ser otimizado, ajustando a composição da solução e da configuração do aparelho eletrofiação, otimizando o alinhamento e morfologia das fibras sendo produzido. Neste protocolo, apresentamos um procedimento para a criação de um aparato electrospinning básico, empiricamente aproximar a concentração entrelaçamento crítico de uma solução de polímero e otimizar o processo de eletrofiação. Além disso, discutimos alguns problemas comuns e técnicas de solução de problemas.

Watch this Scientific Journal Video about Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters at JoVE.com

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Eletrofiação técnicas podem criar uma variedade de andaimes de nanofibras para engenharia de tecidos ou outros aplicativos. Descrevemos aqui um procedimento para otimizar os parâmetros da solução de eletrofiação e aparelhos para obter fibras com a morfologia desejada e alinhamento. Problemas comuns e técnicas de solução também são apresentados.

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. ...

electrospinning-fundamentals-optimizing-solution-apparatus

Research

JoVE Journal

Bioengineering

64.6K visualizações.  University of Michigan. Eletrofiação técnicas podem criar uma variedade de andaimes de nanofibras para engenharia de tecidos ou outros aplicativos. Descrevemos aqui um procedimento para otimizar os parâmetros da solução de eletrofiação e aparelhos para obter fibras com a morfologia desejada e alinhamento. Problemas comuns e técnicas de solução também são apresentados.

Vídeo: Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

Tri-layered Electrospinning para Mimic Arquitetura Arterial nativos usando policaprolactona, elastina e colágeno: um estudo preliminar

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

Bioengenharia

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as oceanography, ecology, biology, and fluid mechanics. Field measurements introduce practical challenges such as environmental conditions, animal availability, and the need for field-compatible measurement techniques. To avoid these challenges, scientists typically use controlled laboratory environments to study animal-fluid interactions. However, it is reasonable to question whether one can extrapolate natural behavior (i.e., that which occurs in the field) from laboratory measurements. Therefore, in situ quantitative flow measurements are needed to accurately describe animal swimming in their natural environment.
We designed a self-contained, portable device that operates independent of any connection to the surface, and can provide quantitative measurements of the flow field surrounding an animal. This apparatus, a self-contained underwater velocimetry apparatus (SCUVA), can be operated by a single scuba diver in depths up to 40 m. Due to the added complexity inherent of field conditions, additional considerations and preparation are required when compared to laboratory measurements. These considerations include, but are not limited to, operator motion, predicting position of swimming targets, available natural suspended particulate, and orientation of SCUVA relative to the flow of interest. The following protocol is intended to address these common field challenges and to maximize measurement success.

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA

This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA.

Medir quantitativamente In situ Fluxos usando um Aparelho Autônomo de Velocimetria Underwater (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as ...

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

Pequenas e Wide Angle X-Ray Scattering Estudos de macromoléculas biológicas em solução

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

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, ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Combinação de Microstereolithography e Electrospinning Produzir Membranas Equipado com nichos para a Regeneração da córnea

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

Fabricando degradáveis hidrogel de Thermoresponsive em várias escalas de comprimento através da extrusão reativa, microfluídica, auto-montagem e eletrofiação

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

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.

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

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 ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

De alta resolução padronização usando dois modos de Jet Electrohydrodynamic: queda na demanda e campo próximo eletrofiação

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

Aparelhos para colheita de tecido Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Three-Dimensional Echocardiographic Method for the Visualization and Assessment of Specific Parameters of the Pulmonary Veins

The dimensions of the pulmonary veins are important parameters when planning pulmonary vein isolation (PVI), especially with the cryoballoon ablation technique. Acknowledging the dimensions and anatomical variations of the pulmonary veins (PVs) may improve the outcome of the intervention. Conventional 2D transoesophageal echocardiography can only provide limited data about the dimensions of the PVs; however, 3D echocardiography can further evaluate relevant diameters and areas of the PVs, as well as their spatial relationship to surrounding structures. In previous literature data, parameters influencing the success rate of PVI have already been identified. These are the left lateral ridge, the intervenous ridge, the ostial area of the PVs and the ovality index of the ostium. Proper imaging of the PVs by 3D echocardiography is a technically challenging method. One crucial step is the collection of images. Three individual transducer positions are necessary to visualize the important structures; these are the left lateral ridge, the ostium of the PVs and the intervenous ridge of the left and right PVs. Next, 3D images are acquired and saved as digital loops. These datasets are cropped, which result in the en face views displaying spatial relationships. This step can also be employed to determine the anatomical variations of the PVs. Finally, multiplanar reconstructions are created to measure each individual parameter of the PVs.
Optimal quality and orientation of the acquired images are paramount for the appropriate assessment of PV anatomy. In the present work, we examined the 3D visibility of the PVs and the suitability of the above method in 80 patients. The aim was to provide a detailed outline of the essential steps and potential pitfalls of PV visualization and assessment with 3D echocardiography.

The dimensions of the pulmonary veins (PV) are important parameters when planning pulmonary vein isolation. 2D transoesophageal echocardiography can only provide limited data about the PVs; however, 3D echocardiography can evaluate relevant diameters and areas of the PVs, as well as their spatial relationship to surrounding structures.

Método Ecocardiográfico Tridimensional para a Visualização e Avaliação de Parâmetros Específicos das Veias Pulmonares

The dimensions of the pulmonary veins are important parameters when planning pulmonary vein isolation (PVI), especially with the cryoballoon ablation ...