Os autores gostariam de agradecer o apoio do New York Região Capital Alliance Research Grant.

1. Bioprinting Plataforma Estabelecimento A modificação de impressora foi baseada em uma impressora jato de tinta térmica HP Deskjet 500 e HP 51626A cartucho de tinta preta. <ol><li> Remova a tampa plástica superior da impressora e cuidadosamente retire o painel de controle a partir da tampa. </li><li> Retire os 3 conexões de cabo entre a parte superior da impressora e base. Remova a parte superior da impressora a partir da base. </li><li> Na parte superior da impressora, remova o pequeno plástico e acessórios de borracha (sistema de limpeza da cabeça de impressão) no lado direito sob o cartucho de tinta. </li><li> Retire a base da bandeja de papel, com molas. </li><li> Remova a placa metálica que cobre a barra de alimentação de papel de plástico. </li><li> Corte a barra de alimentação de papel de plástico na posição da roda de alimentação meio usando uma serra de mão ou outra ferramenta de corte. </li><li> Retire os 2 rodas de alimentação de papel expostos depois da etapa anterior. O plástico roda é muito difícil e um eletrônicoserra será útil. </li><li> Limpe a poeira e detritos usando lenços de ar e etanol enlatados. </li><li> Prenda a parte superior da impressora para a base. </li><li> UV esterilizar a impressora modificada para, pelo menos, 2 horas numa câmara de fluxo laminar antes de usar. </li><li> Corte a tampa de um cartucho de tinta HP 51626A usando um serrote ou outra ferramenta de corte. </li><li> Esvaziar a tinta e remover o filtro que cobre o poço reservatório inferior da cartilagem. </li><li> Lavar o cartucho cuidadosamente, com água corrente. </li><li> Ultrasonicate o cartucho no desionizada (DI) de água durante 10 minutos para remover a tinta residual. </li><li> Examinar o cartucho para certificar-se que toda a tinta foi removida. Lavar ou pulverizar o cartucho cuidadosamente com etanol a 70% para a esterilização, seguido por água DI esterilizado. </li><li> Configurar uma lâmpada ultravioleta de longo comprimento de onda sobre a plataforma de impressão para fornecer capacidade de fotopolimerização simultânea. </li><li> Medir a intensidade UV a uma plataforma da impressão utilizando uma luz UVmetros. Ajustar a distância entre a lâmpada de UV e a plataforma de impressora, de modo a intensidade para o objecto de impressão é entre 4-8 mW / cm2 (cerca de 25 cm de distância da lâmpada para a plataforma de impressora). </li></ol> 2. Bioink Preparação <ol><li> Expansão monocamada de condrócitos <ol><li> Placa 5.000.000 condrócitos humanos em cada balão de cultura de tecidos T175 por expansão das células em Dulbeccos Modified Eagles Médium (DMEM) suplementado com 10% de soro de vitela e 1x penicilina-estreptomicina-glutamina (PSG). As células de cultura a 37 ° C com ar humidificado contendo 5% de CO 2. Altere o meio de cultura a cada 3 dias até que o frasco é de 85% de confluência. Use células da mesma passagem. </li></ol></li><li> Dissolver PEGDA em PBS para uma concentração final de 10% w / v Adicionar fotoiniciador I-2959 a uma concentração final de 0,05% w / v Filtrar esterilizar a solução. </li><li> Suspender condrócitos humanos cultivados na solução PEGDA preparado a 5 x 10 6 células /ml. </li></ol> 3. Cartilagem de tecido de impressão <ol><li> Ligue a impressora e laptop. </li><li> Criar um padrão de impressão de um círculo sólido com 4 mm de diâmetro, utilizando o Microsoft Word ou Adobe Photoshop. <ol><li> Ajuste a posição do padrão e verifique se ele será impresso exatamente no molde plástico. </li><li> Calcule o número de cópias necessárias para atingir a espessura desejada de andaime. Para 4 mm de altura, 220 impressões são necessários para criar o andaime desejado. </li></ol></li><li> Carregue o bioink para o cartucho de tinta. Cubra o cartucho com papel alumínio para proteger da exposição UV direto durante a impressão. </li><li> Enviar comando de impressão para a impressora. Puxe o sensor de papel quando a impressora começa a imprimir. O processo de impressão de todo deve ter menos do que 4 minutos para um andaime com 4 mm de diâmetro e 4 mm de altura. </li><li> Transferência impresso neocartilage para uma placa de 24 cavidades e adicionar 1,5 ml de meio de cultura a cada poço. </li></ol><ol><li> Incubar a neocartilage impresso no VIVO / solução de trabalho MORTOS viabilidade / citotoxicidade em temperatura ambiente por 15 min no escuro. </li><li> Corte o hidrogel célula-laden ao meio e tirar imagens fluorescentes de área de corte. </li><li> Contagem vivo (verde) e as células mortas (vermelho) por um observador cego em cinco imagens tiradas aleatoriamente. Calcular a viabilidade celular através da divisão do número de células vivas pelo número total de células vivas e mortas. </li></ol>

<ol>
	<li>Cui, X., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microvasculature+fabrication+using+thermal+inkjet+printing+technology.">Human microvasculature fabrication using thermal inkjet printing technology.</a> Biomaterials. 30, 6221-6227 (2009).</li><li>Cui, X., Breitenkamp, K., Finn, M. G., Lotz, M., D'Lima, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+human+cartilage+repair+using+thermal+inkjet+printing+technology.">Direct human cartilage repair using thermal inkjet printing technology.</a> Osteoarthritis and Cartilage. 19, (2011).</li><li>Cui, X., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+deposition+of+human+microvascular+endothelial+cells+and+biomaterials+for+human+microvasculature+fabrication+using+inkjet+printing.">Simultaneous deposition of human microvascular endothelial cells and biomaterials for human microvasculature fabrication using inkjet printing.</a> NIP24/digital Fabrication 2008: 24th International Conference on Digital Printing Technologies, Technical Program and Proceedings. 24, 480-483 (2008).</li><li>Cui, X., Dean, D., Ruggeri, Z. M., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+damage+evaluation+of+thermal+inkjet+printed+Chinese+hamster+ovary+cells.">Cell damage evaluation of thermal inkjet printed Chinese hamster ovary cells.</a> Biotechnol. Bioeng. 106, 963-969 (2010).</li><li>Cui, X., Hasegawa, A., Lotz, M., D'Lima, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structured+three-dimensional+co-culture+of+mesenchymal+stem+cells+with+meniscus+cells+promotes+meniscal+phenotype+without+hypertrophy.">Structured three-dimensional co-culture of mesenchymal stem cells with meniscus cells promotes meniscal phenotype without hypertrophy.</a> Biotechnol. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+properties+influence+ECM+production+by+chondrocytes+photoencapsulated+in+poly(ethylene+glycol)+hydrogels.">Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels.</a> Journal of Biomedical Materials Research. 59, 63-72 (2002).</li><li>Elisseeff, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoencapsulation+of+chondrocytes+in+poly(ethylene+oxide)-based+semi-interpenetrating+networks.">Photoencapsulation of chondrocytes in poly(ethylene oxide)-based semi-interpenetrating networks.</a> Journal of Biomedical Materials Research. 51, 164-171 (2000).</li><li>Buskirk, W. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+model+for+cartilage+tissue+engineering+to+regenerate+the+zonal+organization+of+articular+cartilage.">Experimental model for cartilage tissue engineering to regenerate the zonal organization of articular cartilage.</a> Osteoarthritis and Cartilage. 11, 653-664 (2003).</li><li>Sharma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+zonal+organization+into+tissue-engineered+cartilage.">Designing zonal organization into tissue-engineered cartilage.</a> Tissue Engineering. 13, 405-414 (2007).</li><li>Cohen, D. L., Lipton, J. I., Bonassar, L. 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Os autores não têm nenhum interesse financeiro neste estudo.

Este sistema bioprinting 3D com capacidade fotopolimerização simultânea fornece uma resolução de impressão significativamente maior do que o melhor método anteriormente relatado de impressão em in situ de defeitos osteocondrais, utilizando uma seringa extrudido um hidrogel de alginato celular 16. Resolução de impressão mais elevada é particularmente crítica para a engenharia de tecidos de cartilagem para restaurar a cartilagem organização zonal anatómica. Fotopolimerização simultân...

Bioprinting baseada na impressão a jato de tinta térmica é uma das mais promissoras tecnologias de base no campo da engenharia de tecidos e medicina regenerativa. Com controle digital e alto rendimento cabeças fatores células, andaimes e crescimento podem ser precisamente depositado ao desejado bidimensional (2D) e posições (3D) tridimensionais rapidamente. Muitas aplicações de sucesso foram alcançados utilizando esta tecnologia em engenharia de tecidos e medicina regenerativa 1-9. Neste trabalho, uma plataforma bioprinting foi criada com a Hewlett-Packard (HP) Deskjet 500 impressora jato de tinta térmica modificado e um sistema de fotopolimerização simultânea. Hidrogéis sintéticas formuladas a partir de poli (etileno glicol) (PEG) revelaram a capacidade de manter a viabilidade dos condrócitos e promover a produção de ECM condrogénica 10,11. Além disso, a foto-reticulável PEG é altamente solúvel em água, de baixa viscosidade, o que o torna ideal para polímeros usados ​​em simultâneozação durante bioprinting 3D. Neste trabalho, os condrócitos humanos suspensas em poli (etileno) diacrilato glicol (PEGDA; MW 3.400) foram precisamente impresso para construir neocartilage camada por camada, com 1.400 dpi de resolução em 3D. Observou-se distribuição homogênea de células depositadas em um andaime 3D, o que gerou tecido cartilaginoso, com excelentes propriedades mecânicas e produção ECM reforçada. Por contraste, na fabricação manual, as células acumuladas na parte inferior do gel, em vez das suas posições inicialmente depositadas, devido à lenta polimerização andaime, o que levou a formação de cartilagem não homogénea após cultura 2,3.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">HP Deskjet 500 thermal inkjet printer</td>
			<td style="width:221px;">Hewlett-Packard</td>
			<td style="width:195px;">C2106a</td>
			<td style="width:433px;">Discontinued. Purchased refurbished from internet vendor.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">HP black ink cartridge</td>
			<td style="width:221px;">Hewlett-Packard</td>
			<td style="width:195px;">51626a</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Ultraviolet lamp</td>
			<td style="width:221px;">UVP</td>
			<td style="width:195px;">B-100AP</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">UV light meter</td>
			<td style="width:221px;">General Tools</td>
			<td style="width:195px;">UV513AB</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Zeiss LSM 510 laser scanning microscope</td>
			<td style="width:221px;">Carl Zeiss</td>
			<td style="width:195px;">LSM 510</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Dulbeccos Modified Eagles Medium (DMEM)</td>
			<td style="width:221px;">Mediatech</td>
			<td style="width:195px;">10-013</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Penicillin-streptomycin-glutamine (PSG)</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">10378-016</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Accutase cell dissociation reagent</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">A11105-01</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Phosphate buffered saline (PBS)</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">10010-023</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Live/Dead viability/cytotoxicity Kit</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">L-3224</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Poly(ethylene glycol) diacrylate (PEGDA)</td>
			<td style="width:221px;">Glycosan Biosystems</td>
			<td style="width:195px;">GS700</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Irgacure 2959</td>
			<td style="width:221px;">Ciba Specialty Chemicals</td>
			<td style="width:195px;">I-2959</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Human articular chondrocytes</td>
			<td style="width:221px;">Lonza</td>
			<td style="width:195px;">CC-2550</td>
			<td style="width:433px;"></td>
		</tr>
	</tbody>
</table>

human cartilage tissue fabrication using three-dimensional inkjet printing technology

Bioprinting, que está baseada na impressão a jato de tinta térmica, é um dos mais atraentes tecnologias facilitadoras no campo da engenharia de tecidos e medicina regenerativa. Com as células de controlo digitais, os suportes, e os factores de crescimento podem ser depositada precisamente a desejada bidimensional (2D) e os locais (3D) tridimensional rapidamente. Portanto, essa tecnologia é uma abordagem ideal para fabricar tecidos que imitam suas estruturas anatômicas nativas. Para o engenheiro da cartilagem com organização nativa zonal, composição da matriz extracelular (MEC), e as propriedades mecânicas, desenvolvemos uma plataforma bioprinting usando uma impressora jato de tinta comercial com fotopolimerização simultânea capaz de engenharia de tecidos de cartilagem 3D. Condrócitos humanos suspensas em poli (etileno glicol) Diacrilato (PEGDA) foram impressos para a construção neocartilage 3D via montagem camada por camada. As células foram fixadas impressos nas suas posições originais depositados, suportado pelo surroFINANCIAMENTO andaime em fotopolimerização simultânea. As propriedades mecânicas do tecido impresso foi semelhante à cartilagem nativa. Em comparação com a fabricação do tecido convencional, a qual requer a exposição de UV mais, a viabilidade das células impressos com fotopolimerização simultânea foi significativamente maior. Neocartilage impresso demonstraram excelente glicosaminoglicanos (GAG) e a produção de colagénio de tipo II, o que era consistente com a expressão do gene. Portanto, esta plataforma é ideal para distribuição célula precisa e arranjo para a engenharia de tecidos anatômico.

A impressora jato de tinta térmica modificado foi capaz para celular e deposição de andaime no alto rendimento e excelente viabilidade celular. Combinando com fotopolimerização simultânea e biomateriais fotossensíveis, esta tecnologia é capaz de corrigir as células e outras substâncias impressos para os locais inicialmente depositados. De acordo com as propriedades da impressora a jacto de tinta térmica modificada, a resolução de impressão 2D foi 300 dpi com um único volume de gota de tinta de 130 pl. Exi...

Watch this Scientific Journal Video about Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology at JoVE.com

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Os métodos descritos neste trabalho mostram como converter uma impressora jato de tinta comercial em um bioprinter com polimerização UV simultânea. A impressora é capaz de construir a estrutura do tecido 3D com células e biomateriais. O estudo demonstrou aqui construiu uma neocartilage 3D.

Cartilagem Human Tissue Fabricação Usando tridimensional Inkjet Tecnologia de Impressão

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

human-cartilage-tissue-fabrication-using-three-dimensional-inkjet

Research

JoVE Journal

Bioengineering

15.6K visualizações.  Rensselaer Polytechnic Institute.  Os métodos descritos neste trabalho mostram como converter uma impressora jato de tinta comercial em um bioprinter com polimerização UV simultânea. A impressora é capaz de construir a estrutura do tecido 3D com células e biomateriais. O estudo demonstrou aqui construiu uma neocartilage 3D. 

Vídeo: Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impressão de planar e tridimensional de tintas condutoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Bioengenharia

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tridimensional Tecnologia Biomimético: Novel Biorubber Cria Definido micro e macro-escala Arquitecturas em colágeno hidrogéis

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabricação de Inverted coloidal de cristal poli (etileno-glicol) Andaime: Uma Plataforma Cultura celular tridimensional para Liver Engenharia de Tecidos

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Fabricação de dispositivos microfluídicos tridimensional baseados em papel para imunoensaios

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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 ...

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 ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Tridimensional de impressão de materiais termoplásticos para criar automatizado bombas de seringa com controle de gabarito para aplicações microfluídicos

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Fabricação de matriz de cartilagem-derivado Decellularized andaimes

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Bioink extracelular derivado da matriz de tecido sipâncreas para a impressão de construções de tecidopanpático 3D Carregadas de células

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...