Agradecemos ao Dr. Jeffrey Caplan para a sua formação e assessoria em imagem confocal. Agradecemos também o Keck Laboratório de Microscopia Eletrônica e Dr. Chaoying Ni de assistência SEM. Este trabalho é financiado pelo National Institutes of Health (NIDCD, R01DC008965 e R01DC011377). ABZ reconhece NSF Integrativa Pós-Graduação e Pesquisa Estágio programa (IGERT) para financiamento.

1. Assembléia biorreator (Video 1) <ol><li> Fazer um molde de alumínio (matriz circular + pino espaçador) com as dimensões interiores e exteriores pré-determinados (Figura 1). </li><li> Usando o molde a partir do passo 1.1, o fabrico de uma membrana de silicone (diâmetro: 42 mm, espessura: 1,5 mm, Figura 1) com uma manga entranhado (diâmetro: 12 mm, espessura de ~ 0,25 mm, em forma de pino do espaçador da Figura 1) em o meio usando um kit de elastómero de silicone disponíveis comercialmente. </li><li> Faça um par de blocos de acrílico (Figura 2 (4, 5)), com uma abertura circular (diâmetro: 24 mm) no meio, gravar o topo (1,8 cm de espessura) e inferior (0,9 cm de espessura) blocos com sulcos e ranhuras correspondentes 10. </li><li> Sanduíche a membrana de silicone entre os blocos de acrílico emparelhados. Fixe o conjunto com quatro parafusos de canto usando uma chave de fenda de torque micro definido como uma força constante (35 cN · m). Como resultado, uma prova de água, 24 mm de largura e 18 mm de profundidade da câmara de vibração é criada (Figura 2C). </li><li> Monte um &quot;alcance estendido mini-woofer 3 (Figura 2D, 8 Ω/20 W) por baixo da câmara de vibração através de um outro conjunto de parafusos de canto no bloco de acrílico inferior. Neste ponto, um módulo de vibração individual é montado. </li><li> Replicar sete módulos de vibração adicionais. Apor quatro deles a uma das duas barras de alumínio estacionários (40 cm x 10 cm x 2,5 cm), colocando as bases dos altifalantes em furos circulares espaçados uniformemente (diâmetro: 7 cm, espessura: 2 cm) cortado em barras. Estabilizar cada altifalante inserindo um parafuso através do lado da barra de alumínio em cada furo circular. </li><li> Individualmente controlar os alto-falantes por um seletor de alto-falante. Ligue colunas individuais para o selector ligando fios para as entradas positivas e negativas para o corpo do alto-falante, em seguida, para as respectivas saídas do selector. O seletor de alto-falante permite que o sinal de tele gerador de funções, depois de passar por um amplificador de potência, para alcançar todos os oito alto-falantes de uma só vez (Figura 2E). </li><li> Coloque as duas matrizes de câmara, o seletor de alto-falante e componentes eletrônicos associados em um gabinete anti-umidade. Casa todo o conjunto em uma cultura de células incubadora comercial. </li><li> Alimentar os cabos principais (por meio de um tubo de PVC de qualidade médica) que liga o amplificador de potência e selector de coluna através do conjunto de filtro na parte traseira da incubadora. </li></ol> 2. Fabrication andaime e Caracterização <ol><li> Dissolve-se peletes de PCL em clorofórmio a uma concentração de 15% em peso. Carregar a solução numa seringa de 10 ml tapado com uma extremidades cegas agulha 21 G. </li><li> Tranque a seringa em uma bomba de seringa programáveis ​​e definir a taxa de fluxo de 1 ml / h. </li><li> Coloque o coletor de coberta de folha de alumínio através da agulha na horizontal, com uma agulha de distância ponta-de-colecionador de ~ 18 cm. </li><li> Prenda o alli positivoclipe jacaré para o meio da agulha eo jacaré terreno para o coletor de alumínio, em seguida, definir a voltagem da fonte de alimentação de alta tensão de 15 kV. CUIDADO: alta tensão, manter distância da agulha. </li><li> Sequencialmente ligar a bomba de seringa e de alimentação; rapidamente limpo / remover a solução de polímero residual em torno da ponta da agulha com uma toalha de papel seco, antes jactos fibra estável e Taylor cone 19 são formadas. </li><li> Permitir que as fibras se acumulem no colector de Al com uma espessura de ~ 250-300 mM (~ 7 horas sob as condições de fiação corrente). Armazenar os andaimes resultante num exsicador de vácuo durante 1-2 dias para remover qualquer solvente residual. </li><li> Imagem os andaimes, sputter revestido com ouro, utilizando um microscópio eletrônico de varredura para mostrar a morfologia da fibra consistente. 10 </li></ol> 3. Assembléia biorreator e Caracterização <ol><li> Faça um disco cilíndrico (diâmetro: 8 mm) com quatro braços (comprimento: 2mm) para fora do tapete PCL tal como fiados (Figura 2A), em primeiro lugar usando um diâmetro de 12 milímetros biopsia de perfuração para cortar o diâmetro exterior do disco. Em seguida, utilizar um segundo, 8 milímetros biópsia para fazer quatro 2 mm de comprimento entalhes espaçados uniformemente em torno da lâmina circular para marcar onde os braços estão a ser cortados. Depois de marcar com o punção 8 mm, de utilizar uma lâmina de bisturi para cortar as extremidades dos braços para fora. Insira o andaime na ranhura da membrana de silicone via os braços estendidos (Video 1). Achate o andaime inserido pressionando suavemente a superfície, usando uma pinça de cabeça chata. </li><li> Anexar um pequeno pedaço de folha fina Al (8 milímetros x 2 mm, forma ortogonal, Figura 2B) para o cadafalso PCL para auxiliar a reflexão do laser. </li><li> Fixar a membrana de silicone / PCL andaime montado (conforme descrito no passo 1.4) na câmara de vibração. Adicionar 1,5 ml de água na câmara de modo a hidratar o andaime PCL antes vibração. </li><li> Usando o gerador de funções, apresentar sinal de vibraçãoals (por exemplo, 200 Hz ondas sinusoidais com uma tensão de pico-a-pico, Vpp, de 0,1 V) para a câmara de acrílico ensanduichada. Use um voltímetro para medir com precisão a tensão em cada entrada de alto-falante. Nota: a leitura Vpp do gerador de função será diferente da eventual tensão fornecida para o alto-falante. </li><li> Monte o LDV de ponto único e assegurar o sensor laser cabeça de fibra óptica a um tripé cabeça pan-tilt. Ângulo da cabeça do sensor de modo que ele está apontando perpendicular à mesa. Ligue a cabeça do sensor LDV para o módulo de aquisição de dados através de um cabo coaxial, em seguida, o módulo para o laptop via USB. </li><li> Focar o feixe de laser perpendicular em vários pontos predeterminados na membrana de silicone (Figura 2B e Figura 3). </li><li> Utilizando o software de aquisição de dados, registar o deslocamento meados de membrana. Clique em &quot;Configurações de Aquisição&quot; no menu &quot;Opções&quot;; em seguida, alterar o modo de medição para &quot;FFT221;. Em seguida, clique no botão &quot;medição contínua&quot; na barra de ferramentas principal, em seguida, clique no pico que se forma na freqüência escolhida (Figura 6D) para gravar o deslocamento. </li><li> Traça-se a deslocação no meio da membrana normal (w 0) como uma função da posição relativa ao longo do substrato. Construir um mapa de cores em 3D do perfil vibratório superfície usando Origin software de análise de dados de 8,5. </li></ol> 4. Cultura de Células Vibratória <ol><li> MSC derivadas da medula óssea humana de sub-cultura em frascos de cultura de tecido T150 a uma densidade de sementeira inicial de 4.000-5.000 células / cm2 em meio de manutenção MSC. </li><li> Após 7-8 dias de cultura de células (de ~ 85% de confluência), trypsinize as células com um reagente de dissociação de células, tais como Accutase, contagem utilizando um hemocitómetro, centrifugação (440 xg durante 5 min), e re-suspender o sedimento de células em MSC meio de manutenção fresco a uma concentração de 4,5 x 10 6 células / ml. </li><li> Mergulhe o Scaffo PCLld em 70% de etanol S / N. Depois o solvente é evaporado, expor ambos os lados do andaime a luz germicida UV (254 nm) durante 5-8 min. </li><li> Embeber o andaime PCL em uma solução de fibronectina de 20 ug / ml a 37 ° C durante 1 hora. Insira o andaime revestido de fibronectina na membrana de silicone. Montar o bioreactor como detalhado no passo 1. </li><li> Distribuir 40 ul da suspensão celular de forma uniforme sobre o andaime PCL garantido. Permitir que as células se fixem durante 1-1,5 horas antes de se adicionar mais 1,5 ml de meio fresco para a câmara de vibração. </li><li> Cultura cadafalso PCL MSC-carregado estaticamente por 3 dias e atualizar a mídia após a conclusão da cultura estática. </li><li> Impor regimes de vibração selecionados para as construções celulares. Observação: Como um exemplo, as células são sujeitas a um 1-h-on-1-h-off (DO) a 200 Hz vibração com aw de 0 a 40 mM durante 12 horas por dia, durante até 7 dias. As construções submetidas a estímulos vibratórios são designadas como amostras Vib e os blovermelho estaticamente em câmaras de vibração idênticos servir como controles estáticos (Stat). </li></ol> 5. Avaliações biológicas <ol><li> Recolhe 200 ul meios de cultura de células a partir de cada câmara em dias alternados (dias 1, 3, 5, e 7) e juntar as aliquotas da mesma amostra em conjunto (800 mL cada). </li><li> Quantificar a produção celular de metaloproteinase-1 (MMP1) e HA utilizando um kit ELISA MMP1 Desenvolvimento e um kit de ELISA competitivo ácido hialurônico, respectivamente, seguindo procedimento do fabricante. Enquanto isso, ensaiar a produção de precursor de elastina solúvel seguindo o procedimento de ELISA previamente relatado. Oito </li><li> Após a conclusão do último ciclo de vibração no dia 7, remover rapidamente as construções celulares a partir das câmaras de vibração usando pinças cortantes e brevemente lavá-los com fosfato fria salina tamponada (PBS, 4 ° C). </li><li> Para a coloração vivo / morto, incuba-se as construções com iodeto de propídio (1:2000 em PBS) eSyto-13 (1:1000 em PBS), simultaneamente, durante 5 min à TA. Imagem as construções marcadas com um microscópio confocal multiphoton. </li><li> Separadamente, snap-congelar as construções celulares PBS-enxaguados em gelo seco e extrair o ARN celular total, seguindo um protocolo previamente relatado para a análise genética. 9 </li><li> Verificar a quantidade e qualidade do ARN extraído utilizando um espectrofotómetro de UV-Vis. As amostras de ARN com A260/A280 e razões A260/A230 de 1,8-2,2 são utilizados para subsequente análise qPCR. </li><li> Reverso transcrever RNA (500 ng / amostra) em cDNA usando um kit de transcrição reversa disponível comercialmente. </li><li> Realizar a reacção de PCR em um sistema de detecção de sequência em tempo real usando um PCR master mix disponível comercialmente seguindo o procedimento previamente detalhado. Oito </li><li> Analisar os resultados qPCR utilizando software de análise de dados de qPCR comercial. Para garantir a confiabilidade da análise de dados, vários genes de referência (YWHAZ, TBP, PPIA) são empregados como intcontrolos Ernal, e a variância da eficiência de iniciadores específicos é levada em conta. oito </li></ol>

<ol>
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Engenharia de sucesso dos tecidos das pregas vocais funcionais in vitro requer a recriação de um microambiente-como prega vocal para mediar os comportamentos de células multipotentes. É geralmente aceite que o tecido ou órgão estruturas refletem as funções que são necessários para realizar. 22 para os tecidos das pregas vocais, as vibrações de alta frequência que ocorrem durante a fonação são propostas a ser importante para a maturação do tecido. Em nosso estudo, andaimes PCL são u...

A prega vocal humana, composta por uma camada epitelial, a lâmina própria (LP) eo músculo vocal, é um tecido macio especializado que converte o fluxo de ar dos pulmões para ondas acústicas para a produção de som. 1 As pregas vocais oscilam regularmente durante a fonação normal, exibindo as estirpes de até 30% em frequências fundamentais que variam 100-300 Hz. 2 Adulto vocal dobra LP é uma estrutura composta por um gradiente superficial (SLP), de um intermediário (PLI) e um (DLP) camada profunda. Outros grupos de classificação do epitélio eo SLP como a camada mucosa, e combina o ILP e DLP no ligamento vocal. 3 A camada SLP contém principalmente uma matriz amorfa com fibras colágenas pouco dispersos, enquanto que o ligamento é enriquecido com colágeno maduro e fibras de elastina para fornecer a força suficiente. 4 A estrutura e mecânica de pregas vocais nascidos variar significativamente de suas contrapartes adultas. Embora mecanismos que regulam o desenvolvimento das pregas vocais e maturação ainda não são totalmente compreendidos, a evidência experimental apontou para os papéis que definem a tensão mecânica derivada de vocalização. Várias condições médicas, incluindo abuso vocal, infecções, irritantes químicos e procedimentos cirúrgicos, pode danificar a prega vocal. Patologias vocais afeta cerca de 3-9% da população dos EUA. Os métodos atuais de tratamento para patologias vocais são limitados 5 e uma abordagem de engenharia de tecidos à base de células-tronco surgiu como uma estratégia promissora para restaurar a função da prega vocal. Células-tronco mesenquimais (MSCs) são uma alternativa adequada para as primárias de fibroblastos de pregas vocais para engenharia de tecidos das pregas vocais. 6-9 especificação destino de células-tronco e desenvolvimento do tecido subseqüente são mediados pelo nicho específico em que residem, de que o estado mecânico é um fator vital. 10 forças mecânicas são reguladores essenciais do tecido morfogênese umnd homeostase, especialmente para tecidos que são rotineiramente submetidos a uma carga. 11 Do ponto de vista da engenharia de tecidos, tem sido demonstrado que a exposição a estímulos mecânicos fisiologicamente relevantes promove a diferenciação das células-tronco e específico do tecido remodelação da matriz 12-15. Bioreactores de cultura de tecidos são concebidos para simular o ambiente fisiológico pretendido para a célula ou tecido de crescimento in vitro. Para a engenharia de tecidos das pregas vocais, é especialmente crítico para recriar o ambiente mecânico das pregas vocais phonating. Um biorreator ideal prega vocal deve entregar efetivamente sinais vibratórios para células de cultura, permitindo o controle fácil sobre frequência, amplitude e duração das vibrações. Titze e colegas de trabalho elaborou um biorreator prega vocal (T1 biorreator) 16 que combina alongamento estático com alta freqüência (20-200 Hz) oscilações de estimular a produção de proteínas da matriz celular. Using deste biorreactor, Webb e colaboradores 17 estudaram os efeitos de 10-dias, as vibrações de 100 Hz em fibroblastos dérmicos cultivadas num ácido hialurónico (HA), à base de hidrogel. As construções submetidas a vibração exibiu uma expressão elevada de HA-sintase-2 (HAS2), decorina, fibromodulim e metaloproteinase de matriz-1 (MMP1), relativa aos controlos estáticos. Os efeitos estimulantes foram encontrados para ser dependente do tempo. Mais recentemente, o nosso grupo de 18 reuniu um biorreator prega vocal (J1 biorreator) usando um amplificador de potência, um gerador de funções, um alto-falante fechado e uma membrana de silicone em circunferência ancoradas que transfere o ar oscilante para as células unidas. Fibroblastos de prepúcio neonatal cultivadas no biorreactor J1 foram sujeitos a 1 h de vibração a 60, 110 ou 300 Hz, com uma tensão no plano de até 0,05%. Os resultados qPCR sugeriu que a expressão de alguns genes de ECM foi moderadamente alterada em resposta às frequências vibratórias variadase amplitudes. Estes projetos de biorreatores, enquanto intrigante, tem várias limitações. Por exemplo, o sistema de T1 requer um grande número de ligações e barras de acoplamento mecânico, que limita as frequências máximas atingível. Além disso, as células podem ser submetidas a indesejável agitação e fluido perturbação mecânica que complicam a interpretação dos dados. O bioreactor de J1, por outro lado, apresenta relativamente baixa eficiência de conversão de energia e não é fácil de usar. Além disso, a vibração freqüentemente destaca as estruturas celulares em carga da membrana de silicone subjacente. O J2 biorreator prega vocal aqui relatado, projetado com base no mesmo princípio como a versão J1, é otimizado para a consistência e reprodutibilidade. As vibrações que imitam fonação são gerados aerodinamicamente em câmaras de vibração instalados individualmente em poli fibroso MSC-povoada (ε-caprolactona) (PCL) andaimes são effectively garantiu. Laser Doppler Vibrometria (LDV) permite ao usuário verificar o perfil de vibração do conjunto membrana / andaime. Na nossa demonstração, as MSCs são expostos a 200 Hz de vibrações sinusoidais com uma (DE) padrão 1-h-on-1-h-off para um total de 12 hr por dia durante 7 dias. As respostas celulares aos estímulos vibratórios impostas são investigados de forma sistemática. No geral, a prega vocal J2 fornece o maior número de características user-friendly, permitindo estudos de cultura celular dinâmica a ser realizado em uma alta taxa de transferência e moda reprodutível.

<table border="0" cellpadding="0" cellspacing="0" width="853">
	<tbody>
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			<td height="16" style="height:17px;width:248px;">silicone elastomer kit</td>
			<td style="width:188px;">Dow Corning</td>
			<td style="width:124px;">Sylgard 184</td>
			<td style="width:293px;">cure the membrane at 100 C for 2 hr</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCL</td>
			<td>Sigma Aldrich</td>
			<td>440744-500G</td>
			<td>Mn ~ 80 kDa, dissolve overnight</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">chloroform</td>
			<td>Sigma Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">human bone marrow-derived MSCs</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td>received with passage 2</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">MSC maintenance media</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td style="width:293px;">10% FBS in basal media supplemented 
			with L-glutamine, gentamicin and amphotericin</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Accutase cell dissociation reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2006-1MG</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">MMP1 DuoSet ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY901</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HA ELISA kit</td>
			<td>Echelon Biosciences</td>
			<td>&#160;K-1200&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">propidium iodide&#160;</td>
			<td>Life Technologies</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Syto-13&#160;</td>
			<td>Life Technologies</td>
			<td>S7575</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">QuantiTect reverse transcription kit&#160;</td>
			<td>Qiagen</td>
			<td>205311</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">SYBR Green PCR master mix</td>
			<td>Life Technologies</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">replacement speaker</td>
			<td style="width:188px;">DAYTON audio 
			(via Parts Express)</td>
			<td>DS90-8</td>
			<td>paper cone, full range (80-13000 Hz), 85dB</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Ergo Micro torque screwdriver</td>
			<td>Mountz</td>
			<td># 020377</td>
			<td>torque range: 20-120 cN.m</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">stereo speaker selector</td>
			<td>RadioShack</td>
			<td>40-244</td>
			<td>maximum power handling 50 W</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">function generator&#160;</td>
			<td>Agilent&#160;</td>
			<td>33220A</td>
			<td>frequency range 1 &#181;Hz- 20 MHz</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">power amplifier&#160;</td>
			<td>PYLE audio</td>
			<td>PylePro PT2400</td>
			<td style="width:293px;">frequency response: 10 Hz-50 kHz, two speaker 
			channels</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">cell culture incubator&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Steri-Cult 3307</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">syringe pump&#160;</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">High voltage power supply</td>
			<td>Spellman</td>
			<td>CZE 1000R</td>
			<td>output voltage: 0-30 kV</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">scanning electron microscope&#160;</td>
			<td>JEOL-USA</td>
			<td>JSM-7400F</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">desk gold sputter coater</td>
			<td>Denton Vacuum</td>
			<td>DSK00V-0013</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Doppler laser vibrometer&#160;</td>
			<td>Polytec</td>
			<td>PDV-100</td>
			<td>non-contact velocity measurement (0-22 kHz)</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCR sequence detection system&#160;</td>
			<td>Applied Biosystems</td>
			<td>ABI7300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">multiphoton confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510Meta NLO</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">UV-VIS Spectrophotometer&#160;</td>
			<td style="width:188px;">NanoDrop Products 
			via Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">VibSoft Data Acquisition Software</td>
			<td>Polytec</td>
			<td></td>
			<td>acquisition bandwidth up to 40 MHz</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Origin 8.5 data analysis software&#160;</td>
			<td>OriginLab</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">qbasePlus qPCR data analysis software&#160;</td>
			<td>Biogazelle</td>
			<td>V2.3</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">aluminium alloy&#160;</td>
			<td>McMaster-Carr</td>
			<td>Alloy 6061</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">acrylic blocks</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">polycarbonate anti-humidity chamber</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td style="width:293px;">Impact-Resistant&#160;Polycarbonate</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">screws&#160;</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">electronic cable/wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">medical grade PVC tubing</td>
			<td>US Plastic Corp.</td>
			<td>Tygon S-50-HL</td>
			<td>clear, biocompatible</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">10 mL syringe&#160;</td>
			<td>Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">21 G blunt ended needle</td>
			<td>Small Parts</td>
			<td>NE-213PL-25</td>
			<td>1-1/2&#34; length</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Alligator clip adapters&#160;</td>
			<td>RadioShack</td>
			<td>270-354</td>
			<td>fully insulated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">8 mm biopsy punch</td>
			<td><a href="http://www.sklarcorp.com/ProductListUS.aspx?DepartmentID=10&amp;CategoryID=96">Sklar&#160;Surgical Instruments</a></td>
			<td>96-1152</td>
			<td>sterile, disposable</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">12 mm biopsy punch</td>
			<td>Acuderm (via Fisher Scientific)</td>
			<td style="width:124px;">NC9998681</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">tissue culture flasks</td>
			<td>Corning</td>
			<td></td>
			<td>cell culture treated</td>
		</tr>
	</tbody>
</table>

construction and characterization of a novel vocal fold bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

Os andaimes PCL fabricados por electrospinning conter poros intersticiais porte micro e fibras entrelaçadas de forma aleatória, com um diâmetro médio de 4,7 mM (Figura 4A). Numa ampliação maior, ranhuras em nanoescala e poros são visíveis nas fibras individuais (Figura 4B). Revestimento dos andaimes com fibronectina melhora hidrofilicidade e facilita a adesão celular inicial / espalhando no cadafalso PCL (observação não publicada). Formas de onda...

Watch this Scientific Journal Video about Construction and Characterization of a Novel Vocal Fold Bioreactor at JoVE.com

Construction and Characterization of a Novel Vocal Fold Bioreactor

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Construção e Caracterização de um biorreator Fold Novel Vocal

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

construction-and-characterization-of-a-novel-vocal-fold-bioreactor

Research

JoVE Journal

Bioengineering

9.1K visualizações.  University of Delaware. A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Vídeo: Construction and Characterization of a Novel Vocal Fold Bioreactor

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Engenharia Biológica Baseado em enxertos vasculares Usando um biorreator pulsátil

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Bioengenharia

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

Sintéticos, Multi-Layer, auto-oscilante Fabricação Modelo Vocal Fold

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

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  ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Funcionamento de um biorreator de bancada

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigando a separação de fluxo tridimensional induzida por um polip de dobra vocal modelo

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Caracterização de leucócito-rico em plaquetas de fibrina, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Um biorreator Novel de Cultivo de Alta Densidade de diversas comunidades microbianas

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

Construção e configuração de um Bioreactor Fotossintético Algal com Escala de Banco com Monitoramento de Temperatura, Luz e pH para testes de crescimento cinético

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Preparação e caracterização de novas nanopartículas simuladoras de HDL para encapsulação de fatores de crescimento nervoso

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...