Name: tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber
Uploaded: 2016-05-30T15:00:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

Este trabalho foi apoiado por subvenções do NHMRC e Stafford Fox Fundação Médica. Os autores reconhecem a assistência cirúrgica de Sue McKay, Liliana Pepe, Anna Deftereos e Amanda Rixon do Experimental Médica e Unidade Cirúrgica do Hospital de St. Vincent, Melbourne. O suporte também é fornecido pelo Departamento de Inovação, Indústria do Governo do Estado de Victoria e Programa de Apoio à infra-estrutura operacional do Desenvolvimento Regional.

Os protocolos aqui descritos foram aprovados pelo Comitê de Ética Animal de St. Vincent Hospital Melbourne, Austrália, e foram conduzidos em estrita observância às orientações australianos Conselho Nacional de Saúde e Pesquisa Médica. NOTA: Dois protocolos de câmara são descritos abaixo. Os dois modelos diferentes e os seus modelos específicos de câmara são ilustradas na Figura 1. Câmara (1) é feita de policarbonato (para o modelo de rato câmara anelar arteriovenosa). Ele é cilíndrico com um diâmetro interno de 13 mm e altura de 4 mm. Uma janela em um ponto na parede permite o acesso desimpedido para o pedículo. No segundo modelo (por fluxo através de rato modelo de câmara de pedículo), a câmara é feita de poliacrílico e é rectangular (10 x as dimensões de 8 x 4 mm3 internos). Ele tem duas aberturas de 1,5 mm em lados opostos para acomodar a artéria e veia femoral à medida que a câmara de transgredir. 1. Modelo Câmara Rat arteriovenosa Loop (Uma Chamber por animal) NOTA: Antes de iniciar a cirurgia, certifique-se de todos os instrumentos foram devidamente esterilizados. Da mesma forma, garantir o resto instrumentos em toalhas estéreis e estão a uma distância razoável do campo cirúrgico para evitar a contaminação durante o procedimento. <ol><li> Preparação do animal para a cirurgia <ol><li> Use ratos pesando pelo menos 250 g por seu grande tamanho dos vasos para a criação da alça arteriovenosa. </li><li> Anestesiar os animais com 4% inalação de isoflurano. Corroboram profundidade adequada da anestesia, avaliando a ausência de resposta aos pés-pitada. Após a anestesia, o animal manter adequadamente anestesiados durante todo o procedimento com 2% de isoflurano. </li><li> Colocar o animal numa posição supina sobre uma almofada de aquecimento e aplicar lubrificante estéril para os olhos para prevenir a dessecação durante a cirurgia. </li><li> Usando um barbeador elétrico, fazer a barba ambos os virilhas e remover o cabelo com um pedaço de gaze úmida. </li><li> Preparar os locais cirúrgicos com clorexidinasolução de etanol / 70% e armar o animal com toalhas estéreis. Administrar uma dose única de carprofeno (5 mg / kg, subcutaneamente) como analgésico. </li></ol></li><li> Colheita da veia femoral do enxerto <ol><li> Usando uma lâmina # 15, fazer uma incisão na pele de 4 cm de comprimento na virilha esquerdo paralelo ao ligamento inguinal. Isso expõe a almofada de gordura inguinal. </li><li> Cortar a almofada de gordura em circunferência com uma tesoura deixá-lo ligado ao seu pedículo vascular baseado nos vasos epigástricos. </li><li> Usando micro tesouras, libertar os filmy aderências do tecido conjuntivo entre a parede abdominal e os vasos femorais subjacentes. </li><li> Coloque um afastador na parede abdominal e puxe medial. Isso expõe o ligamento inguinal e de todo o comprimento dos vasos femorais. </li><li> Usando micro pinça e tesoura curva dissecar a veia epigástrica e isolá-lo a partir da sua gordura ao redor puxando e corte com cuidado. Esta veia atua como uma corda ao construir o loop. </li><li> using micro fórceps e curvas tesoura micro abrir a bainha perivascular contendo os vasos femorais e nervo todo o caminho desde o ligamento inguinal à sua distal bifurcação para o ramo epigástrica. </li><li> Usando micro fórceps, pegar a veia femoral pela sua adventícia e gentilmente separá-lo dos tecidos circundantes e acompanhando artéria. Faça isso com micro fórceps e curvas tesoura micro apontou-redondas, puxando o tecido distante e cortando-o. NOTA: Não agarrar toda a espessura da parede da veia, pois isso poderia causar trauma para a íntima tornando-se propensos a trombose. </li><li> ramos laterais ligar encontrados durante a dissecção com sutura 10/0 nylon ou coagular-los com um coagulador bipolar. </li><li> Com a veia femoral completamente livre, ligar sua extremidades proximal e distal com suturas de seda 4/0. Certifique-se de obter um enxerto de veia de comprimento, pelo menos, 10 mm e incluem aproximadamente 0,5 cm de comprimento do ramo epigástrica para ser utilizado como um grupo de ancoragem cara cordapara manter a malha aberta na câmara. </li><li> Usando micro fórceps e micro tesoura reta, cortar a adventícia das extremidades do enxerto puxando e corte com cuidado. Isso também pode ser feito mais tarde, antes de anastomoses microcirúrgicas. </li><li> Lave o enxerto de veia com solução salina heparinizada (10 U / ml de heparina) e deixe-a descansar na solução. Fechar a ferida usando corrida contínua 4/0 fio de seda e mais dois ou três pontos isolados simples adicionais. </li></ol></li><li> Criação de arteriovenosa laço e Implantação de Câmara <ol><li> Repita os passos 1.2.1 a 1.2.4 no exatamente da mesma maneira no membro contralateral. </li><li> Usando micro fórceps, dissecar e isolar tanto a artéria epigástrica e veia formar a almofada de gordura ao redor. Para fazer isso, puxando suavemente o tecido longe dos vasos </li><li> Usando micro fórceps, pegar a artéria femoral pela sua adventícia e libertá-la dos tecidos circundantes. Faça isso com micro fórceps e mic pontas round-curvadatesouras ro puxando o tecido distante e cortando-o. Ligadura ou coagular seus ramos laterais. </li><li> Ligar a artéria e veia femoral distai para o surgimento dos vasos epigástricas usando sutura de seda 4/0. </li><li> Colocar uma única braçadeira proximal em cada uma das artéria e veia femorais. Usando um micro tesoura reta afiada, faça um transversal limpo cortado em cada distal navio para a emergência dos ramos epigástrica. Coloque um fundo de contraste de plástico estéril sob os vasos. </li><li> Lave os vasos vigorosamente com generosas quantidades de soro fisiológico heparinizado até que todo o sangue é removido a partir do lúmen. </li><li> Trazer o enxerto de veia no campo operatório e remover qualquer adventícia redundante das extremidades dos navios, como por etapa 1.2.10, se necessário. </li><li> Realizar as duas anastomoses microcirúrgicas com sutura 10/0 nylon. Anastomose da extremidade proximal do enxerto de veia para a veia femoral e a extremidade distai à artéria femoral. Isto irá permitir que o sangue flua fROM arterial para o lado venoso sem resistência de válvulas no interior do enxerto de veia. NOTA: Certifique-se os vasos femorais e o enxerto de veia resto na sua posição natural, sem quaisquer torções. </li><li> Verifique se há vazamentos em ambos os locais de anastomose. Resolver pequenos vazamentos, que parecem não pulsátil de sangue que sai do local da anastomose, colocando um pequeno pedaço de gordura na parte superior e que comprime suavemente durante 5-10 min. Maiores vazamentos pulsantes que rapidamente inundar o campo inteiro vai precisar de pontos adicionais. </li><li> Verifique desobstrução da alça arteriovenosa. oclusão suave da artéria femural deve torná-lo enquanto o mesmo encolher na veia femural deve absorver lo. </li><li> Coloque a base da câmara de engenharia de tecidos sob o loop arteriovenosa com este último na sua posição de repouso natural, sem torções ou torções. </li><li> Fixe a base da câmara ao ligamento inguinal e da fáscia muscular subjacente com 6/0 suturas de nylon. </li><li> Coloque a tampa sobre obase, de modo que os vasos femorais entrar na câmara através de um entalhe (janela no lado da câmara). Ao fechar a tampa, certifique-se que ele pega os ramos epigástricas, entre a base da câmara e tampa, que agem como amarras para segurar a alça arteriovenosa na posição. </li><li> Fechar a ferida usando corrida contínua 4/0 fio de seda e mais dois ou três pontos isolados simples adicionais. </li><li> Permitir ao animal recuperar da anestesia em uma almofada de aquecimento. </li><li> Não deixe o animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. Da mesma forma, não devolva um animal que foi submetido a uma cirurgia para a companhia de outros animais até que esteja totalmente recuperado. 24 horas mais tarde, administrar outra dose única de carprofeno (5 mg / kg, subcutaneamente) como analgésico. </li><li> Tratar a ferida com pomada antibiótica tópica durante 5 dias. Se a ferida é aberta, anestesiar o animal como no passo 1.1.2 1.1.5 através e fechar a ferida como no ponto 1.3.14. Monitorar a saúde de animais diariamente. Eutanásia do animal utilizando uma dose letal de injecção lethabarb intraperitoneal (163 mg / kg, em 0,25 ml de 23 agulha G) se o animal apresentar mais do que um dos sinais moderados inacitivity, falta de apetite, perda de peso e perda de cor. </li></ol></li></ol> 2. flow-through Pedicle Câmara (duas câmaras por animal) <ol><li> Preparação do animal para a cirurgia <ol><li> Repita os passos 1.1.1 até 1.1.4. Duas câmaras pode ser implantado em ambas as regiões inguinais de um único rato. </li></ol></li><li> Isolamento de vasos femorais e Inserção da Câmara <ol><li> Repita os passos 1.2.1 através de 1.2.8. </li><li> Com ambos artéria e veia completamente livre de tecidos circundantes e seus ramos ligados, traga a câmara para o campo operatório. </li><li> Coloque cada um dos vasos femorais intactas na fenda correspondente da base da câmara de certificando-se de que não haja torções ou torções. </li><li> Feche a câmara por attaching a tampa à base. Fechar a ferida usando uma corrida contínua 4/0 sutura de seda mais dois ou três pontos separados adicionais simples e permitir que o animal recuperar, como descrito anteriormente. </li></ol></li></ol> 3. Colheita de Chambers e processamento de tecidos <ol><li> Uma vez que os pontos de tempo do experimento (4-6 semanas pós-implantação) são atingidas, anestesiar o animal como no passo 1.1.2 e repita os passos 1.1.3 através de 1.1.5. </li><li> Abrir a ferida usando uma lâmina de # 15 e a atravessar os tecidos com tesouras até que a câmara está completamente exposta. </li><li> Expor os vasos femorais proximais para a construção e teste de permeabilidade vascular: suavemente ocluir o vaso com duas micropinças, em seguida, o leite do sangue numa direcção distal e finalmente soltar a pinça proximal. Se o navio se enche de sangue novo, isto confirma a permeabilidade. Ligadura os vasos femorais proximalmente no caso de o circuito arteriovenoso e proximal e distalmente no caso de the por escoamento de configuração e eliminar as câmaras com o tecido contendo em bloco. </li><li> No final da experiência, a eutanásia dos animais utilizando uma dose letal de injecção lethobarb intraperitoneal (163 mg / kg em 0,25 ml por 23 L de agulha). </li><li> Fix tecidos em 4% de paraformaldeído à temperatura ambiente durante 24 h. Divide tecidos em várias secções transversais (1-2 mm de espessura) e incorporar em cera de parafina ou composto óptima temperatura de corte de secções de parafina (5 uM) ou secções congeladas (10 fim), respectivamente. 3,4,8,17,22, 24 </li><li> Realizar a coloração histológica de rotina, tais como hematoxilina e eosina para examinar a morfologia geral de tecidos. Execute coloração imuno-histoquímica com anticorpo específico para identificar o tipo de célula de interesse, 3,4,8,17,22,24,29 para a imunocoloração T exemplo troponina em cardiomiócitos. </li></ol>

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K., Cassell, O. C., Kelly, J., Abberton, K. M., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+vascularized+organoid+using+skeletal+muscle+as+the+inductive+source.">Generation of a vascularized organoid using skeletal muscle as the inductive source.</a> FASEB J. 19 (11), 1570-1572 (2005).</li><li>Lim, S. Y., Hern&#225;ndez, D., Dusting, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+vascularized+heart+tissue+from+stem+cells.">Growing vascularized heart tissue from stem cells.</a> J Cardiovasc Pharmacol. 62 (2), 122-129 (2013).</li><li>Poon, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+an+adipogenic+hydrogel+from+subcutaneous+adipose+tissue.">Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.</a> Acta Biomater. 9 (3), 5609-5620 (2013).</li><li>Dilley, R. J., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularisation+to+improve+translational+potential+of+tissue+engineering+systems+for+cardiac+repair.">Vascularisation to improve translational potential of tissue engineering systems for cardiac repair.</a> Int J Biochem Cell Biol. 56, 38-46 (2014).</li><li>Lesman, A., Koffler, J., Atlas, R., Blinder, Y. J., Kam, Z., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vessel-like+networks+within+multicellular+fibrin-based+constructs.">Engineering vessel-like networks within multicellular fibrin-based constructs.</a> Biomaterials. 32 (31), 7856-7869 (2011).</li><li>Hussey, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeding+of+pancreatic+islets+into+prevascularized+tissue+engineering+chambers.">Seeding of pancreatic islets into prevascularized tissue engineering chambers.</a> Tissue Eng Part A. 15 (12), 3823-3833 (2009).</li><li>Chen, X., Aledia, A. S., Popson, S. A., Him, L., Hughes, C. C., George, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+anastomosis+of+endothelial+progenitor+cell-derived+vessels+with+host+vasculature+is+promoted+by+a+high+density+of+cotransplanted+fibroblasts.">Rapid anastomosis of endothelial progenitor cell-derived vessels with host vasculature is promoted by a high density of cotransplanted fibroblasts.</a> Tissue Eng Part A. 16 (2), 585-594 (2010).</li><li>Lin, R. Z., Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor-2+facilitates+rapid+anastomosis+formation+between+bioengineered+human+vascular+networks+and+living+vasculature.">Fibroblast growth factor-2 facilitates rapid anastomosis formation between bioengineered human vascular networks and living vasculature.</a> Methods. 56 (3), 440-451 (2012).</li><li>Dolderer, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+large+volume+adipose+tissue+generation+from+a+vascularized+pedicled+fat+flap+inside+a+chamber+space.">Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space.</a> Tissue Eng. 13 (4), 673-681 (2007).</li><li>Wei, F. C., Lin Tay, S. K., Neligan, P. C., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+techniques+of+microvascular+surgery.">Principles and techniques of microvascular surgery.</a> Plastic Surgery. Volume 1. , 587-620 (2013).</li><li>Sekine, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fabrication+of+functional+three-dimensional+tissues+with+perfusable+blood+vessels.">In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels.</a> Nat.Comm. 4 (1399), 1-10 (2013).</li><li>Lim, S. Y., Sivakumaran, P., Crombie, D. E., Dusting, G. J., P&#233;bay, A., Dilley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichostatin+A+enhances+differentiation+of+human+induced+pluripotent+stem+cells+to+cardiogenic+cells+for+cardiac+tissue+engineering.">Trichostatin A enhances differentiation of human induced pluripotent stem cells to cardiogenic cells for cardiac tissue engineering.</a> Stem Cells Transl Med. 2 (9), 715-725 (2013).</li><li>Lim, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+tissue+engineering+chamber+supports+human+induced+pluripotent+stem+cell+survival+and+rapid+differentiation.">In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.</a> Biochem Biophys Res Commun. 422 (1), 75-79 (2012).</li><li>Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+integration-free+human+induced+pluripotent+stem+cells+from+keratinocytes+by+simple+transfection+of+episomal+vectors.">Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.</a> Stem Cells Transl Med. 3 (7), 787-791 (2014).</li></ol>

Os autores declaram não haver interesses concorrentes.

Engenharia da microcirculação está sendo investigado, essencialmente através de duas abordagens. A primeira envolve o desenvolvimento de uma rede vascular altamente interligada dentro da construção in vitro de modo a que, quando implantados, os capilares do leito vascular hospedeiro ligar com aqueles do transplantado construir através de um processo chamado inosculation, garantindo assim a entrega de nutrientes, não só para a periferia mas também para o núcleo. 21,32,33 Isto é chamado de ...

Fabricação de tecido vascularizado funcional, utilizando uma abordagem de engenharia de tecidos é um paradigma emergente na medicina regenerativa. 1,2 Muitas abordagens para projetar o tecido novo e saudável para a substituição de tecido lesionado ou órgãos defeituosos têm sido desenvolvidos, 3-6 experimentalmente em modelos animais pequenos com promissor potencial clínico 7,8. No entanto, a vascularização continua a ser um dos maiores desafios para a engenharia de tecidos que limitam o seu potencial de crescimento de tecidos tamanho clinicamente relevante. 9 As abordagens actuais para vascularizar tecidos seguir qualquer uma via extrínseca, onde novos vasos crescem a partir do leito vascular destinatário e invadir durante todo o tecido implantado constrói 10 ou uma via de vascularização intrínseca, onde a vasculatura cresce e se expande em uníssono com o tecido acabado de desenvolvimento. 11 A abordagem extrínseca tradicionalmente envolve as células de semeadura em um andaimein vitro e implantando a construção completa para o animal vivo com a expectativa de que os nutrientes, previamente fornecido pelo meio de cultura, será obtida a partir da circulação. 12,13 O conceito é simplista como ingressos vascular é muito lento e apenas implantes muito finas (&lt; 1-2 mm de espessura) permanecerá viável. Fornecendo nutrientes e oxigênio através de uma vascularização suficiente e rápida é o cerne de todas as tentativas de sucesso para crescer substitutos mais complexos e maiores da engenharia de tecidos, como ossos, músculos, gordura e órgãos sólidos. 14,15 vascularização Intrinsic oferece o potencial para construções maiores para desenvolver pelo crescimento tecidual progressiva à altura do seu fornecimento de sangue em expansão. Um projeto é a implantação in vivo em uma câmara de um pedículo vascular, com ou sem um andaime semeado celular. 5,6 Isso abriu o caminho para novos procedimentos para a geração de mais grossos tecidos intrinsecamente vascularizados 16,17. &lt;/ P&gt; Mais recentemente, as estratégias têm sido desenvolvidos para enxertos de tecidos, antes da implantação-vascularizar previamente. Estas redes de vasos sanguíneos incorporados são destinados a unir com os vasos do hospedeiro ao implante permitindo o rápido fornecimento de um suprimento de sangue completo para melhorar a sobrevivência de todas as partes de um enxerto de tecido grosso transplantado 18. Nós pioneira um modelo de engenharia de tecidos in vivo vascularizado em pequenos animais que envolve uma câmara fechada semi-rígida implantadas subcutaneamente contendo um pedículo vascular perfundido e biomateriais contendo as células. A câmara cria um ambiente isquémico que estimula a germinação angiogénica dos vasos implantados. 3 O pedículo vascular pode ser um circuito arteriovenoso reconstruída ou uma artéria de fluxo intacto e veia. 3-6,19 A vasculares couves pediculares um funcionamento e um grande artério rede -capillary-venosa que liga tanto a arteeriole e venosa termina com o pedículo vascular. 3,20 Além disso, a câmara de suporte oco envolvente protege o tecido em desenvolvimento de potencialmente deformando forças mecânicas e prolonga a unidade isquêmico para melhorar a vascularização. 3,21,22 Se o pedículo embarcação é simplesmente implantados em tecido normal e não dentro do espaço protegido da câmara, surgimento angiogênico deixa ao longo da mesma linha do tempo como uma ferida normal e nenhum novo tecido irá acumular em torno do pedículo. Os investigadores usaram esta configuração in vivo para produzir tridimensionais construções de tecido vascularizado funcionais com vasculatura de suporte e de tamanho clinicamente relevante. 4,23 Além disso, as construções de tecido vascularizado artificiais, com a sua pedículo vascular intacta pode ser colhida para transplante subsequente no local da lesão 24,25. Um cenário mais clinicamente viável seria a criação da câmara no local definitivo para a reconstrução sdominam vários como o da mama. Assim, este novo abordagem de engenharia de tecidos pode ter um potencial clínico para proporcionar uma nova fonte de tecido alvo funcional para cirurgia reconstrutiva. 26-28 O protocolo que se segue irá fornecer um guia geral para a construção de uma câmara de engenharia de tecidos vascularizados in vivo no rato, que pode ser adaptada em diferentes modelos animais e utilizados para examinar o intrincado processo de angiogénese, a produção da matriz, e a migração e diferenciação celular.

<table><tbody><tr><td>1 15 Blade Scalpel</td><td>Braun</td><td>BB515</td><td></td></tr><tr><td>1 Toothed Adson Forceps</td><td>Braun</td><td>BD527R</td><td></td></tr><tr><td>1 Needle Holder</td><td>Braun</td><td>BM201R</td><td></td></tr><tr><td>1 Bipolar Coagulator&amp;nbsp;</td><td>Braun</td><td>US335</td><td></td></tr><tr><td>1 Micro Needle Holder B-15-8.3</td><td>S &amp;amp; T</td><td>00763</td><td></td></tr><tr><td>1 Micro Dilator Forceps D-5a.2</td><td>S &amp;amp; T</td><td>00125</td><td></td></tr><tr><td>1 Micro Jeweler&#039;s Forceps JF-5</td><td>S &amp;amp; T</td><td>00108</td><td></td></tr><tr><td>1 Micro Scissors - Straight SAS-11</td><td>S &amp;amp; T</td><td>00098</td><td></td></tr><tr><td>1 Micro Scissors - Curved SDC-11</td><td>S &amp;amp; T</td><td>00090</td><td></td></tr><tr><td>2 Single Clamps B-3</td><td>S &amp;amp; T</td><td>00400</td><td></td></tr><tr><td>2 10/0 nylon suture</td><td>S &amp;amp; T</td><td>03199</td><td></td></tr><tr><td>1 6/0 nylon suture</td><td>Braun</td><td>G2095469</td><td></td></tr><tr><td>2 4/0 Silk Sutures</td><td>Braun</td><td>C0760145</td><td></td></tr><tr><td>Xilocaine 1%</td><td>Dealmed</td><td>150733</td><td>10 mg/ml</td></tr><tr><td>Heparin Sodium</td><td>Dealmed</td><td>272301</td><td>5,000 UI/ml</td></tr><tr><td>Ringer Lactate</td><td>Baxter</td><td>JB2323</td><td>500 ml</td></tr><tr><td>1 dome-shaped tissue engineering chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>1 flow-through chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>Lectin I, Griffonia Simplicifolia&amp;nbsp;</td><td>Vector Laboratories</td><td>B-1105</td><td>1.67 &amp;mu;g/ml</td></tr><tr><td>Troponin T antibody</td><td>Abcam</td><td>Ab8295</td><td>4 &amp;mu;g/ml</td></tr><tr><td>Human-specific Ku80 antibody</td><td>Abcam</td><td>Ab80592</td><td>0.06 &amp;mu;g/ml</td></tr><tr><td>Desmin antibody</td><td>Dako</td><td>M0760</td><td>2.55 &amp;mu;g/ml</td></tr><tr><td>Cell Tracker CM-DiI dye</td><td>Thermo Fisher Scientific</td><td>C-7000</td><td>3 mg/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Lethabarb (sodium pentobarbitone)</td><td><a href="www.virbac.com.au" target="_blank"><img src="/img/new_window.png" /> Virbac Animal Health</a></td><td>LETHA450</td><td>325 mg/ml</td></tr><tr><td>Heat pad flexible</td><td><a href="http://www.redzoneheating.com.au" target="_blank"><img src="/img/new_window.png" /> Redzone Heating</a></td><td>RZ/Medium</td><td></td></tr></tbody></table>

tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber

Na cirurgia de reconstrução, há uma necessidade clínica para uma alternativa aos actuais métodos de reconstrução autólogos, que são complexos, dispendiosos e de comércio um defeito para o outro. A engenharia de tecidos mantém a promessa de abordar esta demanda crescente. No entanto, a maioria das estratégias de engenharia de tecidos não conseguem gerar substitutos de tecidos estáveis ​​e funcionais por causa da má vascularização. Este artigo centra-se em um modelo de câmara in vivo engenharia de tecidos da vascularização intrínseca, onde uma artéria e uma veia perfusão ou como um loop arteriovenosa ou uma configuração pedículo flow-through é dirigido dentro de uma câmara oca protegida. Neste sistema baseado na câmara de germinação ocorre a partir angiogénico nos vasos arteriovenosas e este sistema atrai a migração celular endógeno isquémica e inflamatório accionado que preenche o espaço gradualmente câmara com tecido fibro-vascular. células exógena / implantação matriz no momento da construção da câmara aumenta sur celularvência e determina a especificidade dos tecidos de engenharia que se desenvolvem. Os nossos estudos demonstraram que este modelo de câmara pode gerar com êxito diferentes tecidos, tais como gordura, o músculo cardíaco, fígado e outros. No entanto, são necessárias modificações e refinamentos para assegurar a formação de tecido alvo é consistente e reproduzível. Este artigo descreve um protocolo normalizado para a fabricação de dois modelos de câmara engenharia de tecidos vascularizados diferentes in vivo.

A criação de câmaras microcirúrgico em engenharia de tecido foi realizado como descrito no protocolo acima. Os tecidos geradas dentro das câmaras podem ser examinados histologicamente como descrito no passo 3. protocolo Vários tipos de tecido foram projetados com sucesso utilizando a câmara de vascularizado in vivo (Figura 2). Estas incluem tecido cardíaco com cardiomiócitos neonatais de rato (Figura 2a), o tecido do músculo esqueléti...

Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

tissue-engineering-intrinsic-vascularization-an-vivo-tissue

Research

JoVE Journal

Bioengineering

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

8.8K visualizações.  St Vincent's Institute of Medical Research. This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling. 

Vídeo: Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

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

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A pele completa Defeito Modelo Estudar a vascularização de Biomateriais<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

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. ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

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

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

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

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

Micropatterning and Assembly of 3D Microvessels

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

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

Micropatterning e Montagem de microvasos 3D

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

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

The Loop arteriovenosa (AV) em um modelo animal pequeno para estudar angiogénese e Engenharia de tecido vascularizado

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularizado de microfluidic Bioprinting para a engenharia de tecidos e Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioengenharia Humanos Networks microvascular em camundongos imunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Biologia

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Núcleo/Shell impressão scaffolds para engenharia de tecidos de estruturas tubulares

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Engenharia de Tecidos por Intrínseca Vascularização numa

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