O projeto atual foi financiado pela Universidade de Kansas prova de conceito de fundo. Gostaríamos de agradecer a equipe de enfermagem no KUMC (Kansas City, KS) para nos auxiliar na obtenção de humanos cordões umbilicais e David Jorgensen para ajudar com culturas cochleae.

Todos os procedimentos, incluindo a eutanásia de animais, foram conduzidos de acordo com o cuidado de Animal institucional e uso Comité (IACUC) protocolo aprovado (cálice #2014-2234) da Universidade de Kansas Medical Center (KUMC).
Nota: HWJCs foram isoladas de humanos cordões umbilicais que foram doados pelos pacientes que forneceram consentimento informado e as amostras foram utilizadas em conformidade com os protocolos aprovados pelo Comité de assuntos humanos da Universidade de Kansas (KU-IRB #15402).
1. osso Temporal colheita e isolamento da cóclea
<ol>
	<li>Decapitar um rato apropriadamente sacrificado, 15 semanas cortando entre o crânio e a primeira vértebra cervical com uma afiada tesoura cirúrgica e, em seguida, pulverizar a cabeça com etanol a 70% para desinfetar (figura 1A).</li>
	<li>Bissetriz do crânio em um médio sagital usando o mesmo par afiado de uma tesoura cirúrgica (figura 1B-C, linhas tracejadas).</li>
	<li>Remova o tecido cerebral do crânio utilizando um par de pinças (Figura 1, ponta de seta).</li>
	<li>Identifica o osso temporal através da presença do auditivo e o nervo vestibular raízes (Figura 1E-F, pontas de seta) no interior do crânio e do canal auditivo do exterior (Figura 1F, seta). Corte com cuidado através do crânio para isolar o osso temporal do resto do crânio (Figura 1). Nota: Em alguns casos, pode ser possível intrometer-se delicadamente os circundantes ossos do crânio longe do osso temporal sem a necessidade de corte.</li>
	<li>Insira a pinça bem a abertura do canal auditivo aproximadamente 5 mm (Figura 1 H), então as dicas não arrisque puncionando a cóclea. Suavemente quebrar o osso do bulla (Figura 1 H, top vermelha área sombreada) então ele fraturas (linhaFigura 1I, vermelha pontilhada). Nota: Se necessário, um microscópio de dissecação pode ajudar neste processo para garantir a colocação exata e manipulação de instrumentos.</li>
	<li>Usando a pinça fina, embora bisbilhotar o restante o bulla do osso temporal, expondo o labirinto ósseo da cóclea (Figura 1J, suportes de vermelhos).</li>
	<li>Coloque um prato de Petri grande o suficiente para segurar o osso temporal (por exemplo, 30 mm ou maior) por baixo da dissecação campo de visão do escopo e preenchê-lo com suficiente fosfato tamponado salino (PBS) para cobrir o osso temporal (por exemplo, 0,5 a 1 mL).</li>
	<li>Mergulhe no osso temporal com o bulla removido na PBS com as janelas ovais e redondas da cóclea virada para cima. Nota: A remoção do sistema vestibular da cóclea não é necessária, como o sistema vestibular serve bem como um identificador para a manipulação e orientando recursos da cóclea (Figura 1I e Figura 2B).</li>
	<li>Usando um par de microfuros de fórceps, remova a artéria estapédica, que atravessa o estribo.</li>
	<li>Inserir uma ponta da pinça ultra fina através do arco do estribo onde passou a artéria e delicadamente Levante o estribo para cima. O estribo desarticulado deve levantar fora a janela oval.</li>
	<li>Usando uma ponta da pinça ultra fina, fure o oval e redonda windows.</li>
	<li>Usar um 1X PBS preenchido 28,5 calibre seringa com tubo (diâmetro interno de 0,28 mm, diâmetro externo 0,61 mm) anexada, posicione o tubo sobre a janela oval (Figura 2A -B). Nota: A abertura do tubo pode ser manipulada usando fórceps ultra-fina, ajudando a garantir a colocação exata.<ol>
		<li>Use uma seringa fresca e tubulação para cada cóclea.</li>
	</ol>
	</li>
	<li>Perfundir 2ml de 10%-antibiótico antimicótico (anti-anti) e 10% penicilina-estreptomicina (pen-estreptococos) em PBS através da cóclea mais 5 min para remover a perilinfa. Perfusing muito rápido com muita pressão danificará as belas estruturas dentro da cóclea. Nota: Manualmente, dirigindo a seringa com a mão para perfundir o fluido através da cóclea é eficaz, embora uma bomba de perfusão pode ser usada como uma alternativa.<ol>
		<li>Se perfusing manualmente, use um par de bem, fecho automático fórceps para estabilizar a cóclea (Figura 2A) durante a perfusão, agarrando-se a porção vestibular do osso temporal. Nota: Embora não seja obrigatório, isto deixa ambas as mãos livres para lidar com instrumentos; uma mão pode conduzir a seringa (Figura 2A), enquanto o outro pode posicionar o tubo adequadamente (Figura 2B).</li>
		<li>Desta etapa da frente, tome cuidado para evitar expor a cóclea desnecessariamente ao ar. Introdução de bolhas de ar nos espaços fluidos irá bloquear a circulação do fluido, e mais a secar irá danificar as belas estruturas dentro da cóclea.</li>
	</ol>
	</li>
	<li>Remover e descartar qualquer restantes fragmentos de tecidos e ossos de músculo usando pinça fina.</li>
	<li>Se necessário, armazene cochleae isolado a 4 ° C em anti-anti 10% e 10% solução de caneta-estreptococos em PBS durante sete dias com mudanças regulares de solução antibiótica cada 48 h.</li>
</ol>
2. coclear processamento
<ol>
	<li>Decellularization
	<ol>
		<li>Para cada cóclea, encha um frasco de cintilação de vidro de 20 mL com um 1% de sódio Dodecil sulfato solução (SDS) em água deionizada (DI), que é usada para decellularize a cóclea.</li>
		<li>Corte a ponta de uma pipeta de transferência de 7 mL plástico usando uma lâmina de barbear, de modo que a abertura é grande o suficiente para uma cóclea a passagem.</li>
		<li>Com a pipeta de transferência suavemente elaborar a cóclea na pipeta então só passa por alguns milímetros da borda de corte.</li>
		<li>Expulse da cóclea para o frasco de solução cintilação SDS 1%.</li>
		<li>Circule de 2 mL de SDS 1% em água desionizada de (DI) através da cóclea no frasco de cintilação, com uma pipeta de transferência.</li>
		<li>Coloque o frasco de cintilação em rotacao e permitir que o frasco de cintilação girar a 10 rpm para 72 h à temperatura ambiente.<ol>
			<li>Altere a solução de SDS 1% cada 24 h ao longo de um período de 72 h. Tenha cuidado quando alterar SDS para Nunca exponha a cóclea ao ar.</li>
		</ol>
		</li>
		<li>Lave a cóclea três vezes por 30 min cada com água.</li></ol></li></ol>Executar as lavagens no frasco de cintilação mesma usado para as acusações de SDS; nesta fase, a cóclea é decellularized.
	
	
	<li>Descalcificação
	<ol>
		<li>Remova a água restante do DI o frasco de cintilação, deixando apenas o suficiente para manter a cóclea submergida.</li>
		<li>Para começar a descalcificação, adicione 5 mL de ácido etilenodiaminotetracético (EDTA) (0,02 M) a 10% em água para o frasco de cintilação que contém a cóclea.</li>
		<li>Circule de 2 mL de EDTA 10% em água DI através da cóclea no frasco de cintilação, com uma pipeta de transferência.</li>
		<li>Coloque o frasco de cintilação no rotador e permitir que o frasco de cintilação girar a 10 rpm para 72 h à temperatura ambiente. Altere a solução de EDTA 10% cada 24 h ao longo de um período de 72 h. Tenha cuidado quando alterar soluções para que a cóclea não é exposta ao ar.</li>
		<li>Enxágue a cóclea 3 vezes durante 2 h com PBS 1x; nesta fase, a cóclea é descalcificada.</li>
	</ol>
	</li>
	<li>Armazenamento
	<ol>
		<li>Armazenar os cochleae por até 72 h em uma solução 10% anti-anti, 10% caneta-estreptococos em PBS a 4 ° C. Nota: Os períodos de armazenamento podem ser possíveis com mudanças regulares de solução antibiótica; no entanto, armazenagem prolongada de cochleae transformados não foi validada neste protocolo.</li>
	</ol>
	</li>

3. a colheita e a expansão de hWJCs
<ol>
	<li>Isole o hWJCs de acordo com protocolos previamente publicados22. Brevemente, segmento cordões umbilicais em seções de 3 cm.</li>
	<li>Com cuidado faça uma incisão superficial com um bisturi longitudinalmente em cada segmento de cordão umbilical para desenrolar e expor a geleia de Wharton, do cordão umbilical. Use a pinça para remover os vasos sanguíneos.</li>
	<li>Lave o cordão umbilical segmentos duas vezes com suficiente PBS (por exemplo, 40 mL em uma placa de Petri de 60 mm) para submergir o tecido. Picar finamente segmentos de tecido com um bisturi. Digerir o tecido em uma placa de Petri com 50 mL de meio de digestão de 100mm (colagenase tipo 2 de 0,2%, 1% penicilina-estreptomicina, em baixo-glicose médio (DMEM modificado águia de Dulbecco)).</li>
	<li>Coloque o tecido em digerir médio em um agitador orbital a 50 rpm durante a noite em um 5% CO2, incubadora 37 ° C. No dia seguinte, diluir meio digestão na proporção de 01:16 em 2% antibiótico antimicótico em PBS, pelota e células por centrifugação a 500 x g à temperatura ambiente (~ 27 ° C) por 10 min. Discard sobrenadante.</li>
	<li>Resuspenda pelotas em Mesenchymal Stem Cell crescimento médio (MSCGM) e recombinar as células. Células de placa em uma densidade de 7 x 103 células/cm2 em cultura de tecidos trataram frascos de T-75. Cultura HWJCs em MSCGM e expandir a passagem 5 para experimentos. Nota: HWJCs pode ser sub cultivadas em T-frascos maiores (p. ex., T-150, T-300) quando a passagem para aumentar o rendimento de hWJCs para experimentos.</li>
</ol>
4. infusão de hWJCs em Decellularized Cochleae
<ol>
	<li>Preparar cochleae decellularized
	<ol>
		<li>Utilizando técnica asséptica, lave cochleae armazenado em 1X PBS três vezes para 30 min cada.</li>
		<li>Transferir cochleae em separado poços de uma placa de 24 e adicionar 1 mL de 37 ° C pré aquecido MSCGM.</li>
		<li>Incube cochleae em um 5% CO2, incubadora de cultura de célula de 37 ° C por um período mínimo de 1 h antes da infusão de células.</li>
	</ol>
	</li>
	<li>Dissociar a hWJCs e ressuspender
	<ol>
		<li>HWJCs de lavagem com 37 ° C pré aquecido 1X PBS duas vezes.</li>
		<li>Adicionar suficiente 37 ° C pré aquecido tripsina com 0,5% de EDTA para cobrir a superfície do vaso de célula.</li>
		<li>Incube a hWJCs para até 5 min em um 5% CO2, incubadora de cultura de células de 37 ° C.<ol>
			<li>Verifique-se que 90% das células ter retirado a superfície de cultura de células exibindo células sob um microscópio invertido. Nota: As células que se movem quando o navio de cultura suavemente é aproveitado, tem destacado. As células que permanecem estacionárias, não ter destacado.</li>
			<li>Bata levemente os lados do balão para desalojar mais células anexadas.</li>
		</ol>
		</li>
		<li>HWJCs transferência do navio cultura para um tubo cônico de 50 mL, contendo uma quantidade equivalente de MSCGM para o volume de tripsina utilizando técnica asséptica, usado para dissociar as células.</li>
		<li>O hWJCs de pelotas por centrifugação a 500 x g, à temperatura ambiente (~ 27 ° C) por 5 min.</li>
		<li>Aspire o sobrenadante e ressuspender o hWJCs em MSCGM em uma concentração de 500.000 células/mL.</li>
	</ol>
	</li>
	<li>Perfundir cochleae
	<ol>
		<li>Transferência cochleae para uma nova placa de 24 usando uma pipeta de transferência.</li>
		<li>Adicione uma gota de MSCGM para cada cóclea para impedir qualquer cóclea desseque.</li>
		<li>Delicadamente, Oriente a cóclea usando fórceps ultra fino, de modo que as janelas ovais e redondas estão virada para cima. Nota: Tenha muito cuidado ao manusear diretamente cada cóclea cóclea é facilmente marcado.</li>
		<li>Utilizando uma seringa de insulina estéril de 28,5 mm com tubos conectados, elaborar 0,2 mL resuspended hWJCs (100.000 células).</li>
		<li>Usando a pinça bem esterilizada, posicione tubulação sobre a janela oval.</li>
		<li>Delicada e lentamente perfundir a cóclea com 0,2 mL de ressuspensão hWJCs utilizando uma seringa de insulina 28,5 mm conectada à tubulação de polietileno (diâmetro exterior: 0,61 mm, diâmetro interno: 0,28 mm) ao longo de cerca de 5 min.</li>
		<li>Após a perfusão, adicionar 0,8 mL de 37 ° C pré aquecido MSCGM para o bem que contém a cóclea para trazer o volume total no poço de 1 mL.</li>
		<li>Repita as etapas 4.3.3-4.3.7 para cada cóclea, utilizando uma seringa de fresca e tubos cada vez.</li>
		<li>Lugar perfundidos cochleae em um 5% CO2, incubadora de cultura de células de 37 ° C e mudança mídia três vezes por semana.</li>
	</ol>
	</li>
</ol>
5. preservação e colheita de cóclea
Nota: Cochleae podem ser cultivadas e colhidas em qualquer ponto do tempo de post-perfusão de até 30 dias.
<ol>
	<li>Para preservar os cochleae, corrigir em paraformaldeído 4% em 1X PBS durante a noite a 4 ° C, em uma plataforma de balanço.</li>
	<li>Após a fixação, lave cochleae com PBS 1x, três vezes por 5 min cada.</li>
	<li>Gradualmente, desidratar a cóclea com etanol e limpar com um solvente Hidrocarboneto alifático antes de incorporação em parafina.</li>
	<li>Secção de amostras a uma espessura de 10 µm usando um micrótomo e montar em vidro corrediças do microscópio. Nota: As amostras agora estão prontas para histológica ou imuno-histoquímica de processamento usando protocolos padrão.</li>
</ol>

<ol>
	<li>Raphael, Y., Altschuler, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+innervation+of+the+cochlea.">Structure and innervation of the cochlea.</a> Brain Res Bull. 60 (5-6), 397-422 (2003).</li><li>LeMasurier, M., Gillespie, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair-Cell+Mechanotransduction+and+Cochlear+Amplification.">Hair-Cell Mechanotransduction and Cochlear Amplification.</a> Neuron. 48 (3), 403-415 (2005).</li><li>Neal, C., 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=Hair+cell+counts+in+a+rat+model+of+sound+damage:+Effects+of+tissue+preparation+&amp;+identification+of+regions+of+hair+cell+loss.">Hair cell counts in a rat model of sound damage: Effects of tissue preparation &amp; identification of regions of hair cell loss.</a> Hear Res. 328, 120-132 (2015).</li><li>Ivory, R., Kane, R., Diaz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+hearing+loss:+a+recreational+noise+perspective.">Noise-induced hearing loss: a recreational noise perspective.</a> Curr Opin Otolaryngol Head Neck Surg. 22 (5), 394-398 (2014).</li><li>Stucken, E. Z., Hong, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+hearing+loss:+an+occupational+medicine+perspective.">Noise-induced hearing loss: an occupational medicine perspective.</a> Curr Opin Otolaryngol Head Neck Surg. 22 (5), 388-393 (2014).</li><li>Dille, M. F., 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=Tinnitus+onset+rates+from+chemotherapeutic+agents+and+ototoxic+antibiotics:+results+of+a+large+prospective+study.">Tinnitus onset rates from chemotherapeutic agents and ototoxic antibiotics: results of a large prospective study.</a> J Am Acad Audiol. 21 (6), 409-417 (2010).</li><li>Sajjadi, H., Paparella, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meniere's+disease.">Meniere's disease.</a> Lancet. 372 (9636), 406-414 (2008).</li><li>House, J. W., Brackmann, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinnitus:+surgical+treatment.">Tinnitus: surgical treatment.</a> Ciba Found Symp. 85, 204-216 (1981).</li><li>Kujawa, S. G., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+age-related+hearing+loss+by+early+noise+exposure:+evidence+of+a+misspent+youth.">Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth.</a> J Neurosci. 26 (7), 2115-2123 (2006).</li><li>Ryals, B. M., 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=Avian+species+differences+in+susceptibility+to+noise+exposure.">Avian species differences in susceptibility to noise exposure.</a> Hear Res. 131 (1-2), 71-88 (1999).</li><li>Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D., Hashino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+inner+ear+sensory+epithelia+from+pluripotent+stem+cells+in+3D+culture.">Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture.</a> Nature. 500 (7461), 217-221 (2013).</li><li>Sekiya, T., 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=Cell+transplantation+to+the+auditory+nerve+and+cochlear+duct.">Cell transplantation to the auditory nerve and cochlear duct.</a> Exp Neurol. 198 (1), 12-24 (2006).</li><li>Shi, F., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+for+replacement+of+auditory+neurons+by+stem+cells.">Prospects for replacement of auditory neurons by stem cells.</a> Hear Res. 297, 106-112 (2013).</li><li>Mellott, A. J., Shinogle, H. E., Nelson-Brantley, J. G., Detamore, M. S., Staecker, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+decellularized+cochleae+as+scaffolds+for+inner+ear+tissue+engineering.">Exploiting decellularized cochleae as scaffolds for inner ear tissue engineering.</a> Stem Cell Res Ther. 8, (2017).</li><li>Santi, P. A., Johnson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+ear+tissues+as+scaffolds+for+stem+cell+differentiation.">Decellularized ear tissues as scaffolds for stem cell differentiation.</a> J Assoc Res Otolaryngol. 14 (1), 3-15 (2013).</li><li>Davies, D., Holley, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+alpha+3+and+alpha+6+integrins+in+the+developing+mouse+inner+ear.">Differential expression of alpha 3 and alpha 6 integrins in the developing mouse inner ear.</a> J Comp Neurol. 445 (2), 122-132 (2002).</li><li>Gerchman, E., Hilfer, S. R., Brown, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+extracellular+matrix+in+the+formation+of+the+inner+ear.">Involvement of extracellular matrix in the formation of the inner ear.</a> Dev Dyn. 202 (4), 421-432 (1995).</li><li>Goodyear, R. J., Richardson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrices+associated+with+the+apical+surfaces+of+sensory+epithelia+in+the+inner+ear:+molecular+and+structural+diversity.">Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structural diversity.</a> J Neurobiol. 53 (2), 212-227 (2002).</li><li>Mellott, A. 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=Improving+Viability+and+Transfection+Efficiency+with+Human+Umbilical+Cord+Wharton's+Jelly+Cells+Through+Use+of+a+ROCK+Inhibitor.">Improving Viability and Transfection Efficiency with Human Umbilical Cord Wharton's Jelly Cells Through Use of a ROCK Inhibitor.</a> Cell Reprogram. , (2014).</li><li>Mellott, A. J., Shinogle, H. E., Moore, D. S., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Photo-conversion:+A+second+chance+to+label+unique+cells.">Fluorescent Photo-conversion: A second chance to label unique cells.</a> Cell Mol Bioeng. 8 (1), 187-196 (2015).</li><li>Mitchell, K. E., 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=Matrix+cells+from+Wharton's+jelly+form+neurons+and+glia.">Matrix cells from Wharton's jelly form neurons and glia.</a> Stem Cells. 21 (1), 50-60 (2003).</li><li>Mellott, A. 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=Nonviral+Reprogramming+of+Human+Wharton's+Jelly+Cells+Reveals+Differences+Between+ATOH1+Homologues.">Nonviral Reprogramming of Human Wharton's Jelly Cells Reveals Differences Between ATOH1 Homologues.</a> Tissue Eng Part A. 21 (11-12), 1795-1809 (2015).</li><li>Buytaert, J. A., Johnson, S. B., Dierick, M., Salih, W. H., Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+versus+sTSLIM+3D+imaging+of+the+mouse+cochlea.">MicroCT versus sTSLIM 3D imaging of the mouse cochlea.</a> J Histochem Cytochem. 61 (5), 382-395 (2013).</li><li>Nonoyama, H., 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=Investigation+of+the+ototoxicity+of+gadoteridol+(ProHance)+and+gadodiamide+(Omniscan)+in+mice.">Investigation of the ototoxicity of gadoteridol (ProHance) and gadodiamide (Omniscan) in mice.</a> Acta Otolaryngol. 136 (11), 1091-1096 (2016).</li><li>Dai, C., 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=Rhesus+Cochlear+and+Vestibular+Functions+Are+Preserved+After+Inner+Ear+Injection+of+Saline+Volume+Sufficient+for+Gene+Therapy+Delivery.">Rhesus Cochlear and Vestibular Functions Are Preserved After Inner Ear Injection of Saline Volume Sufficient for Gene Therapy Delivery.</a> J Assoc Res Otolaryngol. , (2017).</li><li>Sutherland, A. J., Converse, G. L., Hopkins, R. A., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioactivity+of+cartilage+extracellular+matrix+in+articular+cartilage+regeneration.">The bioactivity of cartilage extracellular matrix in articular cartilage regeneration.</a> Adv Healthc Mater. 4 (1), 29-39 (2015).</li></ol>

Os autores não têm nada para divulgar.

Temos com sucesso demonstrado que células cocleares nativas podem ser removidas da cóclea através de um processo de decellularization, que permite a utilização da cóclea como um andaime de tecido tridimensional, intrincado. Santi et al. 15 desenvolveu o método inicial para decellularizing cochleae e estimaram com precisão os volumes de muitas estruturas cocleares através com o auxílio de microscopia de luz folha23. Tal trabalho inicial serviu como uma bas...

A cóclea é uma estrutura espiral intrincado encontrada no osso temporal. Ele é composto de um labirinto ósseo exterior e interior, concêntricos labirinto membranoso1. O labirinto membranoso é composto por três espaços fluidos: Scala vestibuli, Scala Scala tympani1e mídia. A mídia scala abriga o epitélio sensorial, que é composto de uma infinidade de tipos de células, mas as células sensoriais de cabelo (HC), que transduce energia mecânica em ondas sonoras para impulsos nervosos2, são de particular interesse. Exposição ao trauma acústico3,4,5, medicação6, doença7,8e envelhecimento9 tudo o que pode resultar em insuficiência auditiva através da morte HC. Perda de cabelo celular em mamíferos é permanente, ao contrário de HCs aviária, que podem se regenerar após lesão10.
Uma variedade de esforços de pesquisa contemporânea procuraram restaurar perdidos HCs, embora as abordagens experimentais específicas variam. Manipulação da expressão do gene no epitélio sensorial e implante de células-tronco diferenciadas fora do corpo são abordagens dominantes no campo, apesar de tem sido métodos de indução que buscam diferenciar as células-tronco em organoids coclear tentativa de11,12,13. Cada uma dessas abordagens é também dependente diretamente as células-tronco, ou as pistas do desenvolvimento, usadas por células-tronco; no entanto, um segundo compartilhados, e potencialmente crítico, o elemento é o ECM da cóclea em si14,15.
O ECM não só fornece suporte físico para as células e tecidos, que inclui uma superfície de adesão celular, sobrevivência, proliferação e migração, mas também tem um papel crítico no desenvolvimento de HCs e a espiral do gânglio15,16 ,17. Naturalmente ocorrendo ECM fornece sinais indutivos que podem orientar a determinação do fenótipo celular e/ou18, de adesão, proliferação e sobrevivência celular. Consequentemente, o uso da cóclea decellularized em combinação com hWJCs cultivadas oferecem uma oportunidade única de explorar o papel da regeneração ECM e HC. HWJCs são um tipo de célula prontamente disponível, sem controvérsias, isolado de humanos cordões umbilicais que se comportar como células-tronco mesenquimais19. HWJCs tem demonstrado a capacidade de diferenciar-se abaixo de20,de linhagens de célula marcante21. Assim, o atual protocolo detalha o isolamento, decellularization e perfusão de cochleae de carcaças de rato C57BL com hWJCs para engenharia de tecidos do ouvido interno.

<table>
	<tbody>
		<tr>
			<td>Allegra X-14R Centrifuge</td>
			<td>Beckman-Coulter</td>
			<td>B08861</td>
			<td></td>
		</tr>
		<tr>
			<td>Intramedic Semi-Rigid Tubing</td>
			<td>Becton Dickinson</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Innova 2000 Orbital Shaler</td>
			<td>Eppendorf</td>
			<td>M1190-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>18155-13</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td>12565271</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>FB087579B</td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 Insulin Syringe</td>
			<td>Fisher Scientific</td>
			<td>14-829-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation Vial</td>
			<td>Fisher Scientific</td>
			<td>03-341-73</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Fisher Scientific</td>
			<td>88-861-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipette</td>
			<td>Fisher Scientific</td>
			<td>22-170-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Fisher Scientific</td>
			<td>15-240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Plate</td>
			<td>Fisher Scientific</td>
			<td>07-200-84</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost PLUS Glass Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipette</td>
			<td>Fisher Scientific</td>
			<td>22-170-404</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Fisher Scientific</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear-Rite 3</td>
			<td>Fisher Scientific</td>
			<td>22-046341</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Forma Series II 3110 Water-Jacekted CO2 Incubator</td>
			<td>Fisher Scientific</td>
			<td>13-998-078</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesenchymal Stem Cell Growth Medium</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Lonza</td>
			<td>CC-3232</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP T-75 Culture Flask</td>
			<td>MidSci</td>
			<td>TP90076</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP T-150 Culture Flask</td>
			<td>MidSci</td>
			<td>TP90151</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP T-300 Culture Flask</td>
			<td>MidSci</td>
			<td>TP90301</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Nikon Instruments</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ts2R-FL Inverted Microscope</td>
			<td>Nikon Instruments</td>
			<td>MFA51010</td>
			<td></td>
		</tr>
		<tr>
			<td>NuAire Class II, Type A2 Biosafety Cabinet</td>
			<td>NuAire</td>
			<td>NU-425-600</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>1% SDS Solution</td>
			<td>Sigma-Aldrich</td>
			<td>436143-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>10% EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for decellularizing mouse cochleae for inner ear tissue engineering

Nos mamíferos, mechanosensory células ciliadas que facilitam a falta de audiência a capacidade de regenerar, que limitou a tratamentos para perda auditiva. Estratégias atuais de medicina regenerativa concentraram-se no transplante de células-tronco ou manipulação genética de em torno de células de suporte na orelha interna para incentivar a substituição de células-tronco danificadas para corrigir a perda de audição. Ainda, a matriz extracelular (ECM) pode desempenhar um papel fundamental na indução e manutenção da função das células de cabelo e não tem sido suficientemente estudada. Usando o coclear ECM como um andaime para cultivar células-tronco adultas pode fornecer insights exclusivos sobre como a composição e arquitetura do ambiente extracelular aids células em sustentar a função auditiva. Aqui nós apresentamos um método para isolar e decellularizing cochleae de ratos para usar como andaimes aceitando perfundidas células-tronco adultas. O protocolo atual, cochleae são isolados da eutanásia de ratos, decellularized e descalcificadas. Depois, células de geleia humana da Wharton (hWJCs) que foram isoladas a partir do cordão umbilical foram cuidadosamente pintadas em cada cóclea. Os cochleae foram usados como biorreatores, e as células foram cultivadas por 30 dias antes de sofrer a transformação para análise. Decellularized cochleae mantidos identificáveis estruturas extracelulares, mas não revelou a presença de células ou fragmentos visíveis de DNA. Células perfundidas na cóclea invadiram a maior parte do interior e exterior da cóclea e cresceram sem incidentes em uma duração de 30 dias. Assim, o método atual pode ser usado para estudar como coclear ECM afeta células desenvolvimento e comportamento.

Usando os métodos apresentados aqui, sucesso decellularization de cochleae foi avaliado examinando-se a presença ou ausência de DNA através de 4', coloração 6-diamidino-2-phenylindole (DAPI). Cochleae foram considerados totalmente decellularized, se o DNA não foi identificado dentro da cóclea decellularized. Uma cóclea nativa de uma experiência anterior que não passam por decellularization ou descalcificação foi usada como controle positivo para ilustrar as estruturas e célu...

Watch this Scientific Journal Video about A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering at JoVE.com

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Um protocolo para Decellularizing Cochleae Mouse para engenharia de tecidos do ouvido interno

O objetivo do presente protocolo é demonstrar um método eficaz para decellularize e descalcificar cochleae rato para utilização como andaimes para aplicações de engenharia de tecidos.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

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Research

JoVE Journal

Bioengineering

10.7K visualizações.  University of Kansas Medical Center. O objetivo do presente protocolo é demonstrar um método eficaz para decellularize e descalcificar cochleae rato para utilização como andaimes para aplicações de engenharia de tecidos.

Vídeo: A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Biologicamente fabricação de materiais derivados injetável para Engenharia de Tecidos do Miocárdio

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Bioengenharia

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Procedimento para a Engenharia de pulmão

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomérico Andaimes em Engenharia de Tecidos Arterial

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Encapsulamento de Cardiomyocytes em um hidrogel de fibrina para engenharia de tecidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Dirigido Cellular Automontagem para Fabricar Anéis Cell-Derived Tecido para Análise Biomecânica e Engenharia de Tecidos

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

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

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

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Força Litografia de Engenharia tecido cardíaco

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protocolo Relativo Avaliação hidrodinâmica de Tri-folheto polímero Válvulas

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Projeto de um biorreator Carregando Mecânica Biaxial para Engenharia de Tecidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...