This work was supported by The British Heart Foundation and The Oak Foundation.

Executar todas as experiências com animais de acordo com protocolos aprovados pelo Comitê Institucional de uso e cuidados de animais de laboratório. 1. Preparação de Meios de Cultura <ol><li> Adicione meios de cultura para a linha de células de fibroblasto humano CCL-153: F-12K Médio, 10% de soro fetal bovino (FBS) e 100 U / ml de penicilina e estreptomicina. </li><li> Adicione reprogramação dos meios para geração de PPCPE: meio de Eagle modificado por KO Dulbecco (DMEM) contendo 20% de substituição KO Soro, 0,1 mM β-mercaptoetanol, 0,1 Mínimo mM Meio Essencial (MEM) não-aminoácidos essenciais, 10 ng / ml do factor de crescimento básico de fibroblastos 2 (bFGF-2) e 100 U / ml de penicilina e estreptomicina. Adicionar bFGF-2 recém cada vez antes de usar a mídia. </li><li> Adicione Diferenciação Meios (DM) para induzir células de músculo liso (SMC) diferenciação: α-MEM meio contendo 10% de FBS, factor de crescimento de 0,1 mM β-mercaptoetanol, 100 U / ml de penicilina e de estreptomicina e 25 ng / ml Derivado de Plaquetas(PDGF-ββ). </li><li> Adicione endotelial Diferenciação Meios (CE-MS) para induzir a diferenciação de células endoteliais (CE): crescimento endotelial meios-2 (EGM-2) meio contendo 50 ng / mL de Factor de Crescimento Endotelial Vascular (VEGF) e 100 U / ml de penicilina e estreptomicina. </li></ol> 2. reprogramação fibroblastos humanos em induzidas Parcialmente células-tronco pluripotentes (PPCPE) <ol><li> Cultura linha de células de fibroblastos humanos CCL-153 em 0,04% de solução de gelatina frascos revestidos em meio de cultura. </li><li> Células de passagem a cada 3 dias, à razão de 1: 6. Use as células antes da passagem 9 para otimizar a eficiência da geração de PPCPE. As células estão prontas para transfecção ao atingir 80 - 90% de confluência. </li><li> Linearizar pCAG2LMKOSimO plasmídeo polycistronic contendo 4 fatores de reprogramação octâmero vinculativo fator de transcrição 4 (OCT4), SOX2, fator Kruppel-like 4 (KLF4) e C-MYC (pCAG-OSKM). Digest 5 ug de plasmídeo com 5 unidades de enzima de restrição Pvul durante 3 horas a 37 ° C. Purificaro plasmídeo com um kit comercial de acordo com o protocolo do fabricante. </li><li> Transfectar 2 x 10 6 de fibroblastos humanos com 4 ug de plasmídeo linearizado pCAG-OSKM por electroporação com o kit de fibroblasto dérmico humano (NHDF) nucleofection de acordo com o protocolo do fabricante. Suavemente semear as células transfectadas para pré 0,04% de gelatina revestida frasco T25 contendo 5 ml de pré-aquecido reprogramação mídia. </li><li> Após 24 h, alterar forma com suplementos de 25 ug / ml de neomicina para seleccionar uma população pura de células transfectadas. </li><li> Alterar médio em dias alternados até dia 4. Nota: fibroblastos humanos tornam-se PPCPE após 4 dias de reprogramação. </li></ol> 3. Diferenciação de PPCPE em endotelial e nas células musculares lisas <ol><li> PPCPE Semente em pratos colágeno IV pré-revestidos contendo DM por 4 dias para induzir a diferenciação em células musculares lisas. Alterar médio em dias alternados até dia 4. </li><li> PPCPE Semente sobre o colágeno IV pré-revestidas pratos containing endotelial-DM durante 6 dias para induzir a diferenciação em células endoteliais. Alterar médio em dias alternados até dia 6. </li></ol> 4. descelularizado Aorta Graft Preparação Nota: Todas as soluções e equipamento deve ser estéril. <ol><li> Sacrifique o mouse por deslocamento cervical e corrigir o mouse em uma posição supina sob dissecção microscópio. </li><li> Corte através do esterno e em torno da caixa torácica para abrir a cavidade torácica. Retirar o coração, pulmões e esôfago para expor a aorta. Com cuidado, retire a gordura peri-aorta com uma pinça. </li><li> Corte a aorta a partir do final anterior com uma tesoura e gentilmente segure a ponta com uma pinça sem corte. Retire a aorta da coluna vertebral para trás por dissecção romba. Feche cuidadosamente todas as artérias se ramificam a partir da aorta por ligação com um electrocoagulador bipolar e corte a partir da extremidade da ligação com uma tesoura. </li><li> Use uma seringa de 5 ml para irrigar o lúmen da aorta com 3 ml de solução salina contendo 100 U de heparina para prevenir a formação de coágulos sanguíneos. </li><li> Corte a extremidade posterior da aorta antes de se ramifica na renal. Manter a integridade da aorta é muito importante durante todo o procedimento. </li><li> Lave o lúmen da aorta ascendente com 5 ml de solução de 0,075% de dodecil sulfato de sódio (SDS), diluída em tampão fosfato salino (PBS). </li><li> Mergulhe a aorta torácica em solução SDS 0,075% em uma placa de Petri de 10 cm. Colocar a placa de Petri num agitador orbital durante 2 horas a 150 rpm à temperatura ambiente. </li><li> Lave o lúmen da aorta com 5 ml de PBS. </li><li> Mergulhe a aorta na PBS em uma placa de Petri de 10 cm. Colocar a placa de Petri num agitador orbital durante 2 horas a 150 rpm à temperatura ambiente. Refresque PBS a cada 20 min. </li><li> Lave o lúmen da aorta com 5 ml de PBS. </li><li> Manter enxerto aorta descelularizado em PBS a 4 ° C durante até uma semana. </li></ol> 5. Dupla Sementeira de PPCPE e Bioreactor Condicionado Nota: Toda a soluçãos e o equipamento deve ser estéril. Realizar todas as operações em uma capa de cultura de tecidos. <ol><li> Montar o circuito de fluxo biorreactor, como ilustrado na Figura 1. Ligue câmara de incubação, meios reservatório, bomba peristáltica, e câmara de conformidade com tubagem. Volume mínimo de meio necessário para a circulação é de 40 ml. </li><li> Pré-condição do navio descelularizado com meio de cultura por imersão da aorta em meio DM em um prato de Petri de 10 cm. Colocar a placa de Petri num agitador orbital durante 1 h, a 50 rpm à temperatura ambiente. </li><li> Inserir um centímetro de comprimento de nylon tubos (OD 0,9 milímetros, 0,75 milímetros ID) em ambas as extremidades do recipiente de descelularização sob microscópio. Amarre a embarcação e os tubos com 8-0 suturas de seda. </li><li> Montar o enxerto descelularizado aorta na câmara de incubação, ligando os tubos de nylon com as portas de entrada e de saída (1/32 &quot;) de tubagem a partir da parede da câmara. Manter a câmara de incubação com 5 ml de meio de cada vez. </li><li> Trypsinize PPCPE adicionando pré-aquecidotripsina para cobrir a placa de cultura e agite levemente o prato 10 vezes. Descarte a tripsina e deixar o prato na incubadora por 2-3 min. Adicionar meio de cultura pré-aquecido no prato e misturar o meio com as células. </li><li> Contar o número de células usando um hemocitômetro. Centrífuga de duas alíquotas de suspensões de células contendo 5 x 10 5 células por cada 5 min a 300 x g. Aspirar o sobrenadante completamente. </li><li> Depois de aspirar o sobrenadante completamente, ressuspender um sedimento dos pips sedimentos celulares em 50 ul de DM. Injetar a suspensão de células no lúmen do vaso descelularizados. </li><li> Em seguida, voltar a suspender o outro pellet pips celular em 100 ul de Matrigel. Pipeta cuidadosamente a mistura sobre o enxerto aorta descelulada. </li><li> Espere por 10-15 min até que a mistura se transforma em um estado de gel-like e uniformemente envolve em torno de superfície externa do navio. Encha a câmara de incubação com DM. </li><li> Colocar todo o biorreactor configuração em um 5% de CO2 a 37 ° C. Rodar manualmente o enxerto descelularizado 90 ° em torno do eixo longitudinal a cada meia hora durante as primeiras 2 horas. </li><li> Mantenha cultura estática durante 12 horas para permitir a adesão celular. </li><li> MS entregar através do lúmen pela bomba peristáltica para induzir a diferenciação de células de músculo liso. Iniciar a taxa de fluxo inicial de 5 ml / min e aumento gradual de 20 mL / min ao longo de 24 h. </li><li> Manter a taxa de fluxo do meio que circula em 20 ml / min durante 24 horas. </li><li> Interromper o fluxo médio e mover a configuração fora da incubadora biorreator. </li><li> Re-semente a luz do enxerto com PPCPE. Trypsinize, contar e centrífuga de 1 x 10 6 PPCPE como nos passos 5,5-5,6. Ressuspender as células em 50 ul de meio EGM-2 contendo 50 ng / ml de VEGF. Injectar a mistura de células para o lúmen do enxerto. </li><li> Alterar a forma da câmara de incubação e toda a circulação de meio EGM-2 que continha 50 ng de VEGF / mL para induzir a diferenciação endotelial. </li><li> Mova o biorreator configuração de volta ao the incubadora. Rodar manualmente o enxerto 90 ° de cada 30 min durante as primeiras 2 horas e, em seguida, manter uma cultura estática, durante 12 h. </li><li> Começar a circular o fluxo de 5 ml / min e aumento gradual de 35 ml / min. Manter a taxa de fluxo a 35 ml / min, durante 5 dias. Alterar circulando e médio câmara a cada dois dias. </li><li> Colher o enxerto de engenharia para posterior enxertia para mouse. </li></ol> 6. Alongamento das Duplo-semeado PPCPE Graft em Ratos <ol><li> Uso NOD.CB17-Prkdc scid / NcrCrl ratos macho como receptores de enxertos navio. Assegure-se sempre que os ratos são cerca de 10 semanas de idade, pesar cerca de 25 - 35 g, e estão em boas condições de saúde. </li><li> Anestesiar rato destinatário através da administração de uma combinação de Hypnorm (25 mg / kg) e Hypnovel (25 mg / kg) por via intraperitoneal. Aplique vaselina pomada oftálmica nos olhos para evitar a secura e sob anestesia. Confirme anesthetization eficiente, garantindo que os ratos têm músculos relaxados e está respirando de forma constante. <ol><li> Corrigir o mouse em decúbito dorsal com o seu pescoço raspada e estendida. Administrar sulfato de atropina (1,7 mg / kg) em combinação com os anestésicos para assegurar que o tracto respiratório do rato permanece claro. </li></ol></li><li> Prepare uma incisão na linha média da mandíbula para esterno do mouse. Sob um microscópio de dissecação com 5- a 10-vezes a amplificação, levantar as glândulas salivares direita lateralmente e remover o músculo cleidomastoid direito para expor a artéria carótida comum direita. </li><li> Suavemente remover tecidos ligados a mobilizar a artéria carótida comum direita a partir da extremidade distai para a extremidade proximal. Ligadura a porção média da artéria carótida comum, duas vezes com uma sutura de seda 8-0 e dissecar entre os dois laços. </li><li> Passe por uma braçadeira feita de tubo de nylon autoclavable em cada extremidade do vaso e corrigir cada extremidade com grampos microhemostat. </li><li> Remover laço de sutura numa extremidade e aplicar uma gota de heparina (100 U / ml). Imediatamente depois, inverter a extremidade distal do the artéria para cobrir todo o corpo cuff e corrigir o navio invertido com uma sutura 8-0 seda para o manguito. <ol><li> Repetir o mesmo procedimento para a outra porção da artéria. Lave artéria com solução salina para remover coágulos sanguíneos. </li></ol></li><li> Implantar um enxerto navio descelulada entre duas extremidades da artéria carótida, deslizando as extremidades dos vasos decelularizadas do ao longo dos punhos das artérias e fixando-o com fio de seda 8-0. Remover pinças vasculares a partir de qualquer termina antes de avaliar a pulsação do enxerto. </li><li> Coloque a glândula salivar de volta à sua posição original. Fechar a pele sobre o local cirúrgico com uma sutura interrompida utilizando um fio de sutura 6-0 de poliglactina. </li><li> Consistentemente observar os sinais vitais do rato após o procedimento for concluído, até que ele retoma a consciência. Sacrifique ratos receptores por luxação cervical e vasos colheita enxertos ou 24 horas ou 3 semanas mais tarde, para posterior análise. </li></ol>

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O protocolo atual indica um som, rápido, estratégia simples, eficiente e reprodutível em que os navios da engenharia de tecidos funcionais podem ser gerados usando PPCPE a partir de fibroblastos humanos. Esta técnica é uma ferramenta valiosa para a medicina regenerativa, engenharia de tecidos e terapia celular específico paciente potencialmente no futuro próximo. As etapas críticas para assegurar a eficácia do protocolo inclui a preparação de PPCPE, preparação de aórticos descelularizados totalmente estér...

A construção de condutas vasculares é uma estratégia fundamental para a reparação cirúrgica de vasos danificados e feridos resultantes de doenças cardiovasculares. Até à data, materiais de enxerto utilizados em cirurgia incluem biocompatível polímeros sintéticos (politetrafluoretileno [Teflon], politetrafluoretileno expandido [ePTFE; Gore-Tex] ou tereftalato de polietileno [Dacron]), enxertos, tecido autólogo (pericárdio ou veia safena) e xenotransplantes 1. Enquanto enxertos artificiais (por exemplo, Gore-Tex e Dacron) são mais comumente usados, estes materiais provável causar inúmeras complicações a curto e longo prazo que incluem estenose, a deposição de cálcio, trombo-embolia e infecções. Embora os pacientes com enxertos biológicos apresentam redução de eventos tromboembólicos, eles ainda se deparam com limitações, como a falência do enxerto secundário e durabilidade encurtado devido à calcificação degradação 2. Portanto, apesar das melhorias significativas na cirúrgico techniques longo dos anos, pesquisadores e clínicos ainda estão sobrecarregados com a necessidade de identificar a conduta ideal para doenças vasculares. Mais recentemente, no campo da engenharia de tecido vascular investigação tem gerado um conceito em que as células são incorporados em suportes biodegradáveis, com o objectivo de criar um ambiente biomimética que simboliza um recipiente funcional de um enxerto bem sucedido. Fundamentalmente, o sucesso das construções vasculares dependem de três componentes essenciais; As células que compreendem o andaime, ou seja, uma camada interior de células endoteliais e uma camada de células de músculo liso, um andaime que contém a matriz extracelular apropriado para proporcionar propriedades mecânicas comparáveis ​​à vasculatura nativa, e a sinalização celular / molecular que é necessário para iniciar / regulação reparação. Dos enxertos a longo prazo e desenvolvimento sustentado dos neo-tecidos são altamente dependentes de semeadura de células eficaz de andaimes, thereby tornar a decisão do tipo de célula de importância crítica. Vários relatos demonstram a utilização de células endoteliais maduras e células do músculo liso a partir de várias fontes para desenvolver condutas de diâmetro pequeno 3-6. Embora promissora, a falta de vasos autólogos suficientes para se obter endoteliais maduras e células musculares lisas continuam a ser um fardo considerável. Mais recentemente, as células estaminais a partir de várias fontes têm sido exploradas para aplicações de engenharia de tecidos vasculares. Células mononucleares de fato, uma grande variedade de tipos de células-tronco, incluindo células-tronco embrionárias 7, células estaminais pluripotentes induzidas (iPSCs) 8,9, PPCPE 10,11, derivadas da medula óssea 12, células-tronco mesenquimais 13, células progenitoras endoteliais e da parede do vaso adulto -derived célula estaminal antigénio-1 (Sca-1) + de células estaminais / progenitoras 14,15 têm sido demonstradas ser capazes de se diferenciarem em células endoteliais ou funcional ou de músculo liso, em resposta a meios definidos econdições de cultura. Além disso, a capacidade de auto-renovação ilimitada das células estaminais torná-los melhores candidatos ao contrário endoteliais maduras e as células musculares lisas, que apenas se podem dividir para um número limitado de vezes antes de se submeter a paragem do crescimento e senescência. A seleção do material andaime para gerar tecido sucesso navio projetado para enxertia depende de vários fatores, tais como biocompatibilidade, propriedades biomecânicas, e taxa de biodegradação. Fundamentalmente, os materiais usados ​​para criar suportes para os enxertos devem ser biodegradáveis ​​e não será montado receptores desnecessária respostas imunes. Além disso, ele deve abranger uma porosidade adequada e microestrutura para a fixação das células e subsequente sobrevivência. Até à data, os materiais mais comuns utilizados para suportes na engenharia de tecido vascular incluem polímeros de ácido poliglicólico, ácido poliláctico, e poli-caprolactona ε 16. Mais recentemente, materiais biológicos têm decelularizadastambém sido aplicada com algum sucesso. Vários laboratórios mostraram que a semeadura vasos porcino descelularizado humano, canino ou com células autólogas proporcionado um enxerto biológico que resistiu a coagulação e a hiperplasia intimal 17-19. Outras estratégias de engenharia de tecido vascular incluem enxertos vasculares extracelulares baseada em proteínas da matriz, por exemplo, células de semeadura em gel de fibrina 13 e folhas de células geradoras sem apoio andaime 20, 21. O actual protocolo demonstra a diferenciação de PPCPE humano em endotelial funcional e células do músculo liso, a geração de um biorreactor que consiste em um recipiente de andaime descelularizado para abrigar células vasculares PPCPE derivados funcionais, e enxerto de vasos em engenharia de tecidos de imunodeficiência combinada severa (SCID ) camundongos. PPCPE são um tipo de célula ideal para usar para engenharia de tecidos de enxertos de vasos porque essas células não formam tumores em camundongos ou aumentar ética eas respostas alo-imunes. Além disso, mostramos que a estratégia para a geração de células musculares pips-células endoteliais e pips-suave é eficiente e reprodutível 10,11. Depois disso, foi elaborado um navio descelulada para a semeadura de células vasculares PPCPE derivados para imitar de perto as proteínas da matriz que existe dentro de um vaso nativo, reforçando assim a enxertia e sobrevivência eficácia. Além disso, a descelularização dos vasos antes da semeadura PPCPE impede a ocorrência de reacções inflamatórias montados por tipos de células imunes tais como os macrófagos. Mais importante, este protocolo não representa apenas uma metodologia para gerar prometendo condutas vasculares para a tradução para os seres humanos, mas também fornece valioso meio de estudar e compreender os mecanismos moleculares que governam a regeneração do tecido vascular através de modelos de mouse.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Human Fibroblasts CCL-153</td>
			<td >ATCC</td>
			<td >CCL-153</td>
			<td>Prenatal human embryonic fibroblasts</td>
		</tr>
		<tr >
			<td >ATCC F-12K Medium (Kaighn&#39;s Modification of Ham&#39;s F-12 Medium)</td>
			<td >ATCC</td>
			<td >30-2004</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ATCC</td>
			<td >30-2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout DMEM medium optimized for embryonic stem cells</td>
			<td >Life technologies (Gibco)</td>
			<td >12660-012</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout Serum Replacement</td>
			<td >Life technologies (Invitrogen)</td>
			<td >10828-028</td>
			<td></td>
		</tr>
		<tr >
			<td >Human Basic FGF-2&#160;</td>
			<td >Miltenyi Biotech</td>
			<td >130-093-837</td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Life technologies (Invitrogen)</td>
			<td >32571093</td>
			<td></td>
		</tr>
		<tr >
			<td >Human PDGF</td>
			<td >R&#38;D System</td>
			<td >120-HD-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Gelatin Solution 2%</td>
			<td >Sigma</td>
			<td >G1393</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td>
			<td >Addgene</td>
			<td >20866</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;PvuI Restriction Enzyme</td>
			<td >New England Biolabs</td>
			<td >RO150S</td>
			<td></td>
		</tr>
		<tr >
			<td >SureClean Plus</td>
			<td >Bioline</td>
			<td >BIO-37047</td>
			<td></td>
		</tr>
		<tr >
			<td >Nucelofection Kit (NHDF Kit)</td>
			<td >LONZA</td>
			<td >VPD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Neomycin</td>
			<td >SIGMA</td>
			<td >G418</td>
			<td>Selection of&#160;</td>
		</tr>
		<tr >
			<td >KL 1500 LCD, Illumination for Stereo Microscopy</td>
			<td >SCHOTT</td>
			<td >KL 1500 LCD</td>
			<td>Cold light illumination for stereo microscopy</td>
		</tr>
		<tr >
			<td >Nikon Zoom Steromicroscope SMZ800</td>
			<td >Nikon</td>
			<td >SMZ800</td>
			<td></td>
		</tr>
		<tr >
			<td >Heparin sodium salt</td>
			<td >Sigma</td>
			<td >H3393</td>
			<td></td>
		</tr>
		<tr >
			<td >10% SDS Stock Solution Molecular Biology Reagent</td>
			<td >Severn Biotech</td>
			<td >CAS 151-21-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td >Sigma</td>
			<td >D8537</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrigel (10mg/ml)</td>
			<td >BD</td>
			<td >A6661</td>
			<td></td>
		</tr>
		<tr >
			<td >Shaker IKA Vibrax with Shaking platform VX 7&#160;</td>
			<td >Jepson Bolton&#39;s, Janke&#38;Kunkel</td>
			<td >S32-102</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S Digital Pump Drive</td>
			<td >Cole-Parmer</td>
			<td >WZ-07523-80</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S 6-channel, 6-roller cartridge pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S large cartridges for pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-75</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td>
			<td >Cole-Parmer&#160;</td>
			<td >WZ-96410-14</td>
			<td >Tubing goes through the peristaltic pump</td>
		</tr>
		<tr >
			<td >0.5mm ID, 0.8 mm OD Silicone Tubing</td>
			<td >SILEX</td>
			<td >N/A</td>
			<td>Tubings connect incubation chamber, media reservoir and compliance chamber&#160;</td>
		</tr>
		<tr >
			<td >Fitting Reducer 0.5 to 1.6, natural Polypropyline</td>
			<td >Ibidi</td>
			<td >10829</td>
			<td>Adapter connect above two types of tubings</td>
		</tr>
		<tr >
			<td >1/32&#34; Tubing, ID 0.01&#34; (250&#181;m) Material: PEEK</td>
			<td >LabSmith</td>
			<td >T-132-010P</td>
			<td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td>
		</tr>
		<tr >
			<td >One-Piece Fittings&#160;</td>
			<td >LabSmith</td>
			<td >T-132-100</td>
			<td>Fix the above tubings through the incubation chamber wall</td>
		</tr>
		<tr >
			<td >Nylon tubes (OD 0.9mm, ID 0.75mm)&#160;</td>
			<td >Smiths Medical</td>
			<td >N/A</td>
			<td>Tubings insert into two ends of the aorta graft</td>
		</tr>
		<tr >
			<td >NOD.CB17-Prkdcscid/NcrCrl mouse</td>
			<td >Charles River</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 8-0&#160; silk</td>
			<td >ETHICON</td>
			<td >W819</td>
			<td ></td>
		</tr>
		<tr >
			<td >Hypnorm</td>
			<td >Vetapharm</td>
			<td >Vm21757/4000</td>
			<td >Neuroleptanalgesic for use in mice</td>
		</tr>
		<tr >
			<td >Hypnovel (Midazolam)</td>
			<td >Roche</td>
			<td >59467-70-8</td>
			<td >Induction of anaesthesia</td>
		</tr>
		<tr >
			<td >Dissecting microscope</td>
			<td >Carl Zeiss</td>
			<td >Stemi 2000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nylon Tubing</td>
			<td >Portex LTD</td>
			<td >800/200/100/200</td>
			<td >0.65 mm in diameter and 1 mm in length; to make artery cuff</td>
		</tr>
		<tr >
			<td >Electrocoagulator</td>
			<td >Martin&#160;</td>
			<td >SN 54.131</td>
			<td >Ligation of artery branches on aorta</td>
		</tr>
		<tr >
			<td >Bipolar micro hemostat forceps</td>
			<td >Martin</td>
			<td >80-91-12-04</td>
			<td >Fixation of vessel ends</td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFX-7</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFL-3dZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >D-5aZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini applier&#160;</td>
			<td >AESCULAP</td>
			<td >FE572K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micro hemostats clips</td>
			<td >AESCULAP</td>
			<td >FE720K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 6-0 VICRYL</td>
			<td >ETHICON</td>
			<td >V489</td>
			<td ></td>
		</tr>
	</tbody>
</table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

A geração bem sucedida de PPCPE foi confirmada 4 dias após nucleofecting fibroblastos humanos com um plasmídeo linearizado pCAG2LMKOSimO transportando quatro fatores de transcrição, Oct4, SOX2, KLF4 e c-Myc (OSKM). PPCPE apresentaram um fenótipo marcadamente distinta quando comparados com os fibroblastos (Figura 2A) e expressa os quatro factores de reprogramação no ARNm (Figura 2B) e de proteína (Figura 2C) 10 níveis. A eficácia de um enxerto vasc...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Aqui, apresentamos um protocolo para gerar engenharia de tecidos enxertos de vasos que são funcionais para enxerto em camundongos por semeadura dupla parcialmente célula-tronco pluripotentes induzidas (PPCPE) - derivados de células musculares lisas e PPCPE - células endoteliais derivadas em um biorreator descelulada navio andaime.

Geração e Alongamento das embarcações da engenharia de tecidos em um modelo de rato

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

generation-and-grafting-of-tissue-engineered-vessels-in-a-mouse-model

Research

JoVE Journal

Bioengineering

11.9K visualizações.  King's College London BHF Centre.  Aqui, apresentamos um protocolo para gerar engenharia de tecidos enxertos de vasos que são funcionais para enxerto em camundongos por semeadura dupla parcialmente célula-tronco pluripotentes induzidas (PPCPE) - derivados de células musculares lisas e PPCPE - células endoteliais derivadas em um biorreator descelulada navio andaime. 

Vídeo: Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

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 ...

Bioengenharia

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

Preparação de Membranas de Luz respondem por um enxerto de superfície combinada e Processo Postmodification

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Técnica Cirúrgica para a Implantação de engenharia de tecidos enxertos vasculares e Subsequente<em&gt; In Vivo</em&gt; Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

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

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

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

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

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

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produção, Caracterização e usos potenciais de uma mucosa esofágica Modelo 3D Human Tissue-engenharia

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

A Tissue Combinada 3D Engineered<em&gt; In Vitro</em&gt; /<em&gt; In Silico</em&gt; Modelo de tumor de pulmão para prever a eficácia da droga em fundos específicos de mutação

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

aplicações de<em&gt; In Vivo</em&gt; Teste Funcional do Rato tibial anterior para a Avaliação da engenharia de tecidos Repair Músculo Esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Engenharia de Tecido Tridimensional de Redes de Astrócitos Alinhadas para Recapitular Mecanismos de Desenvolvimento e Facilitar a Regeneração do Sistema Nervoso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Uma nova técnica de sutura tenorrhaphy com enxerto de colágeno projetado por tecido para reparar defeitos grandes do tendão

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

A propagação e a implantação de um enxerto traqueal biossintética engenharia de tecido em um modelo do rato

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...