Este trabalho é apoiado financeiramente pelo NIH (Grants 1R01AR072027-01, 1R03AR069383-01), NSF Career Award (1653702) e Universidade de Connecticut.

1. Síntese de JBNTs
NOTA: O monômero JBNT foi preparado conforme publicado anteriormente11.
<ol><li>Síntese do composto A1<ol>
<li>Prepare uma solução contendo 8,50 g de ácido 2-cianoacético e 9,80 g de etilcarbama em 25 mL de tolueno e 2,5 mL de N, N-dimetilformamida. Adicione 4,90 mL de cloreto de fósforo dropwise. Em seguida, aqueça a mistura a 70 °C e continue mexendo por 1,5 h.</li>
		<li>Esfrie a mistura de reação à temperatura ambiente e despeje 100 g de água gelada. Extrair a camada aquosa com acetato etílico (3 x 250 mL) e lavar com 100 mL de salmoura. Seque a camada orgânica sobre sulfato de sódio anidro, filtrar e evaporar voláteis sob vácuo para obter um sólido amarelo-pálido.</li>
		<li>Sujeito o sólido amarelo à cromatografia da coluna flash de sílica (3% de metanol/diclorometano) para produzir 15,61 g de composto A1 como pó branco.</li>
	</ol></li>
	<li>Síntese do composto A2<ol>
<li>Misture 3,12 g de composto A1 e 2,75 g de carbonato de potássio em 50 mL de N, N-dimetilformamida. Mexa em temperatura ambiente por 2h.</li>
		<li>Adicione 2,6 mL de dissulfeto de carbono à suspensão de reação. Continue mexendo por 4h.</li>
		<li>Adicione etanol absoluto à mistura de reação a 0 °C. Filtre o amarelo precipitado e lave com éter dietil. Seque sob vácuo durante a noite para produzir 6,18 g de composto A2 como sólido amarelo-pálido.</li>
	</ol></li>
	<li>Síntese do composto A3<ol>
<li>Adicione 2,7 mL de iodeto de metila em 15 mL de acetonitrilo. Além disso, prepare separadamente uma solução de composto A2 adicionando 6,18 g de A2 a 80 mL de água/acetonitrilo (proporção 7:3). Misture as soluções e mexa à temperatura ambiente por 30 minutos.</li>
		<li>Aqueça a mistura de reação a 95 °C e continue mexendo por 3h.</li>
		<li>Esfrie a mistura de reação à temperatura ambiente e, em seguida, evaporar os voláteis sob vácuo.</li>
		<li>Extrair o resíduo 3x com 100 mL de acetato de etila e lavar com salmoura. Seque a camada orgânica sobre sulfato de sódio anidro, filtre e evapore os voláteis sob vácuo para obter um sólido amarelo.</li>
		<li>Sujeito o sólido amarelo à cromatografia da coluna flash de gel de sílica (30% de acetato/hexanos etílicos) para produzir 4,52 g do composto A3 como um sólido amarelo-claro.</li>
	</ol></li>
	<li>Síntese do composto A4<ol>
<li>Prepare a solução de alilamina adicionando 925 μL de alilamina em 20 mL de etanol. Adicione esta solução dropwise à solução do composto A3, preparado adicionando 3,14 g de A3 em 75 mL de etanol absoluto, mais de 30 min. Em seguida, aqueça a mistura de reação ao refluxo por 16 h.</li>
		<li>Esfrie a mistura de reação à temperatura ambiente e evapore os voláteis para dar um sólido amarelo.</li>
		<li>Sujeito o sólido amarelo à cromatografia da coluna flash de sílica (50% de acetato/hexanos etílico a 2% de metanol/diclorometano) para produzir 1,80 g de composto A4 como cristal branco.</li>
	</ol></li>
	<li>Síntese do composto A5<ol>
<li>Adicione 2,05 g de cloridrato de guanidinium e 7,8 mL de etoóxido de sódio (21 wt% no etanol) em 14 mL de etanol absoluto. Aqueça a mistura a 45 °C por 15 min.</li>
		<li>Filtre o material insolúvel e adicione o filtrado diretamente na solução do composto A4, preparado adicionando 2,70 g de A4 em 40 mL de etanol absoluto. Aqueça a mistura de reação ao refluxo por 16 h.</li>
		<li>Filtre o precipitado para obter 2,65 g de composto A5 como um sólido off-white.</li>
	</ol></li>
	<li>Síntese do composto A6<ol>
<li>Adicione 24,9 mL de trietilamina lentamente à mistura de chorume obtida adicionando 2,11 g de composto A5 e 1,10 g de 4-Dimethylaminopyridina em 120 mL de tetrahidrofurano acima de 1 min. Mexa em temperatura ambiente por 2 minutos.</li>
		<li>Adicione 20,7 mL de di-tert-butyl dicarbonato dropwise à mistura de reação e continue mexendo à temperatura ambiente por 40 h.</li>
		<li>Adicione 20 mL de água para saciar a reação. Evaporar os voláteis sob vácuo.</li>
		<li>Extrair o resíduo viscoso vermelho com 500 mL de acetato de etila. Em seguida, lave-o com 250 mL de água, 75 mL de ácido cítrico 10%, 2x com 200 mL de água, 200 mL de bicarbonato de sódio saturado e 200 mL de salmoura. Seque a camada orgânica sobre sulfato de sódio anidro, filtre e evapore os voláteis sob vácuo para dar um sólido vermelho/laranja.</li>
		<li>Sujeito o sólido à cromatografia da coluna flash de gel de sílica (0-20% acetato/hexanos) para produzir 3,87 g de composto A6 como espuma branca.</li>
	</ol></li>
	<li>Síntese do composto A7<ol>
<li>Adicione N-metilmorpholine N-óxido (50 wt.% em água, 1,64 mL) dropwise à solução do composto A6 (2,93 g) em acetona/água (8:1, 58,5 mL) e mexa à temperatura ambiente por 5 minutos.</li>
		<li>Adicione tetraóxido de ósmio (solução aquosa de 4%) à mistura durante um período de 3 minutos e continue mexendo à temperatura ambiente por 24h.</li>
		<li>Sacia a reação com sulfito de sódio aquoso de 1,0 M até que a solução fique incolor. Remova os voláteis sob vácuo para resultar em um chorume branco na água. Extrair o resíduo com 350 mL de diclorometano e depois lavar com 150 mL de água seguido de uma lavagem com 150 mL de salmoura.</li>
		<li>Seque a camada orgânica sobre sulfato de sódio anidro, filtre e evapore os voláteis sob vácuo para produzir 2,45 g de composto A7 como um sólido branco.</li>
	</ol></li>
	<li>Síntese do composto A8<ol>
<li>Adicione 760 g de periodide de sódio à mistura de 1,36 g de composto A7 em 35 mL de diclorometano/água (6:1, 35 mL) e mexa à temperatura ambiente por 42 h.</li>
		<li>Filtre o material insolúvel e concentre o filtrado sob vácuo. Extrair o resíduo resultante com 250 mL de diclorometano e, em seguida, lavar com 100 mL de água e 100 mL de salmoura. Seque a camada orgânica sobre sulfato de sódio anidro, filtre e evapore os voláteis sob vácuo para dar o produto bruto.</li>
		<li>Sujeito o produto bruto à cromatografia da coluna flash de sílica (5-40% acetato/hexanos etílicos, depois 5% metanol/diclorometano) para produzir 1,08 g de composto A8.</li>
	</ol></li>
	<li>Síntese do composto A9<ol>
<li>Prepare uma solução de 830 mg de composto A8 em 15 mL de 1,2-dicloroetano. Adicione a solução de dropwise à mistura de 6-benzyloxycarbonylamino-2-L-amino-hexanoico ácido trimetilsilyl etil ester (649,5 mgs) e N, N-diisopropiltilamina (591 μL) em 24 mL de 1,2-dicloroetano ao longo de 3 min e mexa em temperatura ambiente por 15 minutos.</li>
		<li>Adicione 360,3 mg de triacetoxyboroidride de sódio à solução e continue mexendo à temperatura ambiente por 24 h.</li>
		<li>Sacie a mistura de reação com 6 mL de água. Extrair a mistura de reação 3x com 200 mL de diclorometano e lavar com 200 mL de ácido cítrico de 10% em água, 2x com 200 mL de água, 200 mL de bicarbonato de sódio saturado e 200 mL de salmoura nessa ordem. Seque a camada orgânica sobre sulfato de sódio anidro, filtre e evapore os voláteis sob vácuo para obter o produto bruto.</li>
		<li>Sujeito o produto bruto à cromatografia da coluna flash de gel de sílica (5-30% acetato/hexanos) para produzir 885 mg composto A9.</li>
	</ol></li>
	<li>Síntese do monômero JBNT<ol>
<li>Dissolva 0,54 g de composto A9 (0,54 g) em 10 mL de solução de ácido/tioanisola trifluoroacética de 94% e mexa à temperatura ambiente por 72 h.</li>
		<li>Adicione éter dietil (80 mL) à mistura de reação e centrífugue o precipitado para baixo. Despeje o ácido trifluoroacético claro contendo supernanato e lave o precipitado branco com éter dietilo e metanol para produzir 168,6 mg de monômero JBNT bruto(Arquivo Suplementar 1).</li>
		<li>Purifique o monômero JBNT bruto por uma cromatografia líquida de alto desempenho (HPLC) com uma coluna de fase inversa. O programa de isolamento está listado abaixo: Solvente A: 100% água; solvente B: 100% acetonitrilo; solvente C: pH=1 Solução hcl de água; fluxo: 3 mL/min (ver Tabela 1).</li>
		<li>Colete o maior pico em cerca de 7,2 min.</li>
	</ol></li>
</ol>2. Fabricação para JBNT/FN
<ol><li>Adicione 80 μL de solução aquosa JBNT de 1 mg/mL a 40 μL de solução aquosa de 1 mg/mL FN e pipeta várias vezes.</li>
	<li>Verifique a presença de suspensão branca sólida após a pipetação por cerca de 10 s. NOTA: Um vídeo foi filmado para capturar o processo de automontagem do NM (Arquivo Suplementar 2).</li>
</ol>3. Observação de espécimes liofilizados
NOTA: Esta etapa é realizada para mostrar a estrutura do andaime de NM feita de JBNTs e FN.
<ol><li>Prepare soluções FN, JBNT e FN/JBNT NM para observação de materiais liofilizados. Diluir 10 μL de 100 μg/mL de FN com 40 μL de água destilada para fazer uma solução de 20 μg/mL de FN.</li>
	<li>Prepare a solução de 100 mg/mL de JBNTs diluindo 5 μL de 1 mg/mL JBNTs em 45 μL de água DI.</li>
	<li>Misture 10 μL de 100 μg/mL FN e 5 μL de 1 mg/mL de JBNTs em 35 μL de água DI para formar a solução NM.</li>
	<li>Cubra três poços de microplacas de fundo redondos de 96 bem claras com a solução FN, JBNT e solução NM, respectivamente. Cada poço recebe 50 μL da solução.</li>
	<li>Realizar a liofilização, como descrito abaixo, para visualizar a estrutura do andaime do NM.<ol>
<li>Congele a placa a -80 °C durante a noite.</li>
		<li>Ligue o instrumento liofilizado e a bomba de vácuo.</li>
		<li>Pressione o botão de desligar para reduzir a temperatura do sistema para -50 °C.</li>
		<li>Coloque a placa congelada no instrumento liofilizado e feche todas as aberturas dos instrumentos.</li>
		<li>Clique no botão iniciar para reduzir a pressão do sistema para 0,9 mbar.</li>
		<li>Depois de 4h, pressione o botão de aerado e abra a válvula para aumentar a pressão do sistema à pressão atmosférica.</li>
		<li>Tire a placa do instrumento liofilizado.</li>
		<li>Use um microscópio para observar o FN, JBNTs e o NM auto-montado resultante. Tire várias fotos com uma câmera para os materiais.</li>
	</ol>
</li>
</ol>4. Medição de espectro de absorção
NOTA: Use a alteração de espectro para FN e JBNTs para apresentar o auto-montado JBNT/FN NM12.
<ol><li>Misture 30 μL de 100 μg/mL FN com 15 μL de água destilada e 5 μL de 1 mg/mL JBNTs para preparar a solução JBNT/FN NM. A suspensão sólida branca pode ser vista depois de pipetting a solução várias vezes. NOTA: As concentrações finais de JBNTs e FN na solução são de 100 μg/mL e 60 μg/mL, respectivamente. JBNTs e soluções FN na mesma concentração à utilizada na solução NM também foram preparadas como soluções de controle.</li>
	<li>Diluir 5 μL de 1 mg/mL de JBNTs com 45 μL de água destilada para preparar a solução de controle JBNT.</li>
	<li>Diluir 30 μL de 100 μg/mL FN com 20 μL de água destilada para preparar a solução de controle FN.</li>
	<li>Meça o espectro de absorção UV-vis das soluções FN, JBNT e JBNT/FN NM com o espectotômetro (ver Tabela de Materiais) para caracterizar a montagem de NM.<ol>
<li>Clique no botão UV-Vis. Limpe a superfície de teste do espectrofotômetro e solte 2 μL de água destilada sobre ele. Meça-o como em branco de 190-850 nm.</li>
		<li>Limpe a superfície de teste do espectrofotômetro e solte 2 μL de solução FN sobre ele. Meça o espectro de absorção UV-Vis da solução FN de 190 nm a 850 nm.</li>
		<li>Limpe a superfície de teste do espectrofotômetro e solte 2 μL de solução JBNT sobre ele. Meça o espectro de absorção da solução JBNT de 190 nm a 850 nm.</li>
		<li>Limpe a superfície de teste do espectrofotômetro e solte 2 μL da solução JBNT/FN NM sobre ele. Meça os espectros de absorção da solução JBNT/FN NM de 190 nm a 850 nm.</li>
	</ol>
</li>
</ol>5. Preparação do JBNT/FN NM para microscopia eletrônica de transmissão (TEM)
<ol><li>Limpe várias grades antes da coloração negativa com um limpador de plasma básico (ver Tabela de Materiais).</li>
	<li>Realize a coloração negativa para os espécimes seguindo dois processos diferentes.<ol>
<li>Solte 3 μL de 1 mg/mL de solução aquosa JBNTs e 3 μL de solução JBNT/FN NM em grades separadas e deixe-a por 2 minutos. Em seguida, enxágüe cada grade com 100 μL de solução de acetato de urânl (UA) de 0,5%. Escorra a solução em excesso com papel filtro e o ar seque as grades.</li>
		<li>Misture 9 μL de solução JBNT/FN NM com 3 μL de solução UA de 2,0% e pipeta várias vezes. Pipeta a solução mista nas grades e mantenha-a nas grades por 2 minutos. Use um papel filtro para remover a solução mista das grades e secar as grades à temperatura ambiente.</li>
	</ol>
</li>
	<li>Por fim, caracterize a amostra para JBNTs, JBNT/FN NM manchada com a etapa 5.2.1, e JBNT/FN NM manchada com a etapa 5.2.2 com o TEM.</li>
</ol>6. Ensaio de função biológica in vitro
<ol><li>Adicione 200 μL de controles negativos (NC, PBS), JBNTs, FN e as soluções JBNT/FN NM em um poço de um slide de câmara de 8 poços, respectivamente.</li>
	<li>Congele a tampa de 8 poços com o instrumento liofilizado para revestir materiais na parte inferior dos poços. O protocolo de secagem congelante é o mesmo do passo 3.5.</li>
	<li>Em seguida, adicione 10.000 células/poços hMSCs (Passagem 3) nos poços e incuba-os por 24h a 37 °C com 5% de CO2.</li>
	<li>Após a incubação, pipeta o meio de cultura celular dos poços e enxágue a cultura com solução 1x PBS.</li>
	<li>Adicione 100 μL da solução fixativa a cada poço com células e deixe por 5 minutos. Enxágüe as células suavemente com 1x PBS duas vezes.</li>
	<li>Diluir 1% Triton-X 100 a 0,1%. Adicione 100 μL de 0,1% Triton-X 100 a cada poço com células e deixe por 10 minutos. Enxágüe as células suavemente com 1x PBS duas vezes.</li>
	<li>Diluir a falooidina de Rhodamina para 1,65 μM. Adicione 100 μL de Rhodamine Phalloidin a cada poço com células e incubar o vidro de cobertura de câmara mantendo-se da luz à temperatura ambiente por 30 minutos. Enxágüe as células suavemente com 1x PBS duas vezes.</li>
	<li>Capture as imagens celulares com um microscópio fluorescente.</li>
	<li>Conte os números de células em imagens fluorescentes para cada grupo. Calcule a densidade de adesão celular por uma média de três áreas aleatórias em cada poço.</li>
	<li>Apresentar todos os dados como desvio padrão ± médio (SD). Realizar análises estatísticas utilizando-se um teste t-cauda-de-cauda, seguido da análise de variância (ANOVA) com p<0,05, que é considerado estatisticamente significante.</li>
</ol>

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V., Khorsand, B., Geary, S. M., Salem, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Printing+of+scaffolds+for+tissue+regeneration+applications.">3D Printing of scaffolds for tissue regeneration applications.</a> Advanced Healthcare Materials. 4 (12), 1742-1762 (2015).</li><li>Shi, W., 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=Structurally+and+functionally+optimized+silk-fibroin-gelatin+scaffold+using+3D+printing+to+repair+cartilage+injury+in+vitro+and+in+vivo.">Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo.</a> Advanced Material. 29, 1701089 (2017).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. G., Fenniri, H., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+inflammatory+activation+by+self-assembling+Rosette+nanotubes+in+human+Calu-3+pulmonary+epithelial+cells.">Low inflammatory activation by self-assembling Rosette nanotubes in human Calu-3 pulmonary epithelial cells.</a> Small. 4 (6), 817-823 (2008).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. G., Fenniri, H., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosette+nanotubes+show+low+acute+pulmonary+toxicity+in+vivo.">Rosette nanotubes show low acute pulmonary toxicity in vivo.</a> International Journal of Nanomedicine. 3 (3), 373-383 (2008).</li></ol>

Dr. Yupeng Chen é um co-fundador da NanoDe Therapeutics, Inc.

Neste estudo, desenvolvemos um NM biomimético auto-montado, que foi formado com JBNTs inspirados em DNA e FN. Ao preparar a solução JBNT, o pó liofilizado JBNT deve ser dissolvido na água em vez de PBS, pois a PBS causará aglomeração de JBNTs, o que inibe sua montagem. Além disso, o NM também deve ser montado em água se quisermos observar as estruturas nanofibrilas do NM, pois o sal em PBS vai empacotar com fibras NM, o que pode reduzir muito a resolução das imagens.
O NM tem most...

As células-tronco mesenquimais humanas (hMSCs) têm mostrado o potencial de auto-renovação e auto-diferenciação ao longo de diferentes linhagens mesenquimais, o que ajuda na regeneração e manutenção dos tecidos1. Com base no potencial de diferenciação, os HMSCs são considerados candidatos a lesões de tecido mesenquimal e terapia de transtorno hematopoiético2. hMSCs têm mostrado a capacidade de promover a cicatrização de feridas aumentando a reparação de tecidos, angiogênese e reduzindo a inflamação3. No entanto, sem assistência bioquímica ou biomaterial, a eficiência para que os hMSCs atinjam um tecido alvo e funcionem no local desejado é baixa4. Embora vários andaimes projetados tenham sido utilizados para atrair hMSCs para aderir às lesões, alguns locais como a fratura da placa de crescimento, no meio de um osso longo, não são facilmente acessíveis pelos andaimes convencionais pré-fabricados, que podem não se encaixar perfeitamente em um local lesionado de forma irregular.
Aqui, desenvolvemos um nanomaterial biomimético que pode se auto-montar in situ e ser injetado em uma área alvo de difícil acesso. O bioescato injetável NM é composto por nanotubos base janus (JBNTs) e fibronectina (FN). Os JBNTs, também conhecidos como Nanotubos Rosette (RNTs), são derivados de pares de bases de DNA, especificamente timina e adenina, aqui5,6,7. Como visto na Figura 1,os nanotubos são formados quando seis moléculas da base de DNA derivada se auto-montam através de ligações de hidrogênio para formar um plano6. Seis moléculas são então empilhadas uma sobre a outra em um plano através de uma forte interação pi-empilhamento7, que pode ser de até 200-300 μm de comprimento. Os JBNTs são projetados para imitar morfologicamente fibras de colágeno para que a FN reaja com elas.
FN é uma glicoproteína adesiva de alto peso molecular, que pode ser encontrada na matriz extracelular (ECM)9. Estes podem mediar a fixação de células-tronco a outros componentes do ECM, particularmente o colágeno10. Projetamos JBNTs para imitar morfologicamente fibras de colágeno para que a FN possa reagir com eles para formar NM em poucos segundos através de ligação não covalente. Portanto, nm é um promissor bioescato a ser injetado em um local de fratura óssea que não poderia ser acessível pelos andaimes convencionalmente fabricados. Aqui, o NM injetável apresenta uma excelente capacidade de melhorar a ancoragem hMSC in vitro, exibindo seu potencial para servir como um andaime para regeneração tecidual.

<table>
	<tbody>
		<tr>
			<td>1,2-dichloroethane</td>
			<td>Alfa Aesar</td>
			<td>39121</td>
			<td></td>
		</tr>
		<tr>
			<td>2-cyanoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>C88505</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Dimethylaminopyridine</td>
			<td>TCI America</td>
			<td>D1450</td>
			<td></td>
		</tr>
		<tr>
			<td>8 wells Chambered Coverglass</td>
			<td>Thermo Fisher</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>absolute ethanol</td>
			<td>Thermo Fisher</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34851</td>
			<td></td>
		</tr>
		<tr>
			<td>allylamine</td>
			<td>Sigma-Aldrich</td>
			<td>145831</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC32G</td>
			<td></td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>concentrated hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>Thermo Fisher</td>
			<td>15230147</td>
			<td></td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-tert-butyl dicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>361941</td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylcarbamate</td>
			<td>Sigma-Aldrich</td>
			<td>U2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermo Fisher</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixative Solution (4 % formaldehyde prepared in PBS)</td>
			<td>Thermo Fisher</td>
			<td>R37814</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidinium hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>A13543</td>
			<td></td>
		</tr>
		<tr>
			<td>hexanes</td>
			<td>Sigma-Aldrich</td>
			<td>227064</td>
			<td></td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl iodide</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine</td>
			<td>Alfa Aesar</td>
			<td>A17114</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methylmorpholine N-oxide</td>
			<td>Alfa Aesar</td>
			<td>A19802</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetraoxide</td>
			<td>Alfa Aesar</td>
			<td>45385</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Thermo Fisher</td>
			<td>20012050</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphoryl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>201170</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>347825</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse phase column</td>
			<td>Thermo Fisher</td>
			<td>25305-154630</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>silica gel</td>
			<td>TCI America</td>
			<td>S0821</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium ethoxide</td>
			<td>Alfa Aesar</td>
			<td>L13083</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium periodide</td>
			<td>Sigma-Aldrich</td>
			<td>71859</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>S0505</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium triacetoxyborohydride</td>
			<td>Alfa Aesar</td>
			<td>B22060</td>
			<td></td>
		</tr>
		<tr>
			<td>spectrophotometer(NanoDrop One/One&#7580; UV-Vis)</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Growth Medium BulletKit</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrahydrofuran</td>
			<td>Sigma-Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>thioanisole</td>
			<td>Sigma-Aldrich</td>
			<td>T28002</td>
			<td></td>
		</tr>
		<tr>
			<td>toluene</td>
			<td>Sigma-Aldrich</td>
			<td>179418</td>
			<td></td>
		</tr>
		<tr>
			<td>triethylamine</td>
			<td>Alfa Aesar</td>
			<td>A12646</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Alfa Aesar</td>
			<td>A12198</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher</td>
			<td>HFH10</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Thermo Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion

Um NM biomimético foi desenvolvido para servir como um andaime biológico de engenharia tecidual, que pode melhorar a ancoragem de células-tronco. O NM biomimético é formado a partir de JBNTs e FN através de auto-montagem em uma solução aquosa. Os JBNTs medem 200-300 μm de comprimento com canais ocos hidrofóbicos internos e superfícies hidrofílicas externas. Os JBNTs são carregados positivamente e as FNs são cobradas negativamente. Portanto, quando injetados em uma solução aquosa neutra, eles são unidos através de laços não covalentes para formar os feixes NM. O processo de automontagem é concluído dentro de alguns segundos sem qualquer iniciador químico, fonte de calor ou luz UV. Quando o pH da solução NM for inferior ao ponto isoelétrico das FNs (pI 5.5-6.0), os feixes de NM serão auto-liberados devido à presença de FN carregado positivamente.
NM é conhecido por imitar a matriz extracelular (ECM) morfologicamente e, portanto, pode ser usado como um andaime injetável, que fornece uma excelente plataforma para melhorar a adesão do HMSC. A análise da densidade celular e os experimentos de imagem de fluorescência indicaram que os NMs aumentaram significativamente a ancoragem de hMSCs em comparação com o controle negativo.

Nossos estudos descobriram que a formação do NM de JBNTs e FN é rápida, o que aconteceu em 10 segundos. Como mostrado na Figura 2,o flocule branco foi obtido quando a solução JBNT foi misturada com a solução FN e pipetada várias vezes. O processo de formação de NM é completamente biomimético. Não são necessários estímulos externos. O processo de fabricação é muito mais fácil do que o de alguns biomateriais emergentes, que são baseados em luz ultravioleta ou iniciador qu...

Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

O objetivo deste protocolo é mostrar a montagem de uma nanomatrix biomimética (NM) com nanotubos base janus (JBNTs) e fibronectina (FN). Quando co-cultivados com células-tronco mesenquimais humanas (hMSCs), os NMs apresentam excelente bioatividade no incentivo à adesão dos HMSCs.

Fabricação de uma Nano-Matriz Biomimética com Nanotubos base janus e fibronectina para adesão de células-tronco

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

fabrication-biomimetic-nano-matrix-with-janus-base-nanotubes

Research

JoVE Journal

Bioengineering

4.0K visualizações.  University of Connecticut. O objetivo deste protocolo é mostrar a montagem de uma nanomatrix biomimética (NM) com nanotubos base janus (JBNTs) e fibronectina (FN). Quando co-cultivados com células-tronco mesenquimais humanas (hMSCs), os NMs apresentam excelente bioatividade no incentivo à adesão dos HMSCs.

Vídeo: Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabricação microfluida de fibras poliméricas e biohibridas com tamanho e forma predesigned

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Bioengenharia

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Helicoidal Organização dos Fatores de Coagulação Sanguínea VIII em Lipid Nanotubos

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Usando impedância Cell-substrato e imagens de células vivas para medir as mudanças em tempo real no celular Adesão e De-adesão Induzidas por Matrix Modificação

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

Fabricação de uma funcionalizados Magnetic bacteriana Nanocellulose com óxido de ferro nanopartículas

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication pelo Solvent-assistida Lipid bicamada (SALB) Método

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e Montagem de microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubos de DNA como uma ferramenta versátil para estudar semiflexível polímeros

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabricação e validação de um sistema de órgãos-em-microplaqueta com eléctrodos integrados para quantificar diretamente a resistência elétrica de Transendothelial

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabricação de poços de Agarose personalizado para a propagação de células e tecido anel Self-assembly usando moldes 3D-impresso

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Metodologia para neuromodulação Biomimetic químico de Retinas de ratos com o neurotransmissor glutamato em Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...