Este trabalho foi apoiado financeiramente por projetos NSFC (81522023, 81530059, 91639113, 81772000, 81371699 e 81401534).

O uso de animais experimentais foi aprovado pela Animal experiências éticas Comitê de Nankai University e realizado em conformidade com o guia para o cuidado e uso de animais de laboratório.
1. fabricação de enxertos Electrospun PCL
Nota: Neste documento, uma técnica de eletrofiação foi utilizada para fabricar os enxertos vasculares.
<ol><li>Preparar soluções PCL de 25% em peso e 10% em peso, dissolvendo PCL em uma mistura de metanol e clorofórmio, respectivamente, (relação de volume de 1:5), à temperatura ambiente (RT) para 12 h.</li>
	<li>Carrega a solução PCL em uma seringa de vidro de 10 mL.</li>
	<li>Coloque a seringa com uma agulha 21-G.</li>
	<li>Coloca o mandril de aço inoxidável (2 mm de diâmetro, 25 cm de comprimento) sobre o instrumento de coleta.</li>
	<li>Para enxertos de fibra mais grossa, use a solução PCL de 25% em peso, a distância de trabalho de 17 cm da ponta da agulha para colecionador, taxa de fluxo de 8 mL/h e tensão de 11 kV como eletrofiação parâmetros. Para enxertos de diluente-fibra, use a solução PCL de 10% em peso, distância de trabalho de 20 cm da ponta da agulha para colecionador, uma taxa de fluxo de 2ml/h e a tensão de 18 kV como eletrofiação parâmetros.</li>
	<li>Certifique-se que os enxertos obtidos são colocados no vácuo durante a noite para remover o solvente residual. Esterilizar todos os instrumentos antes do procedimento e manter técnica asséptica durante todo.</li>
	<li>Antes da implantação, desinfecte os enxertos por imersão-los em 10 mL de etanol a 75% por 30 min e expô-los à luz UV durante a noite.</li>
	<li>Medições de tamanho da fibra e dos poros: calcular o diâmetro da fibra média usando software ImageJ baseado na digitalização de imagens de microscopia eletrônica de varredura (MEV).</li>
	<li>Teste mecânico de andaimes:<ol>
<li>Corte os andaimes tubulares em seções de 3 mm de comprimento, usando uma lâmina de barbear. Medir a espessura de andaimes usando um micrômetro.</li>
		<li>Coloque os andaimes tubulares em uma máquina de ensaio de tração com capacidade de carga de 100 N.</li>
		<li>Fixe os andaimes com uma distância de 1 mm grampo inter e puxar longitudinalmente a uma taxa de 10 mm/min até ruptura. Medir a resistência à tração e alongamento final na ruptura. Calcular o módulo de Young da região linear inicial (até 5% de tensão) da curva tensão-deformação.</li>
	</ol>
</li>
</ol>2. modelo de implantação do rato Aorta Abdominal
Nota: Todos os materiais e instrumentos utilizados na cirurgia são estéreis. Durante a cirurgia, certifique-se que o operador usa uma máscara de gaze e luvas estéreis para evitar infecções. Assegure que a temperatura é mantida a 27-30 ° C para manter a temperatura do corpo animal. Siga orientações IACUC locais sobre analgesia.
<ol><li>Use ratos Sprague Dawley masculinos pesando 240-270 g como destinatários da prótese vascular. Certifique-se do que rato tem jejuado 24 h antes da cirurgia. O objectivo dos ratos jejum por 24 h é vazio as fezes no trato intestinal suficientemente, assim, ampliam o horizonte do operador.</li>
	<li>Segure o pescoço costas do rato e mantenha sua cabeça para baixo, insira a agulha de springe na cavidade abdominal do abdômen inferior. Induzi o rato para anestesia com hidrato de cloral (330 mg/kg) por uma injeção intraperitoneal.</li>
	<li>Confirme anesthetization adequada, garantindo que o rato tem relaxado os músculos e respiração firme. Coloque o rato sob o microscópio de operação na posição supina.</li>
	<li>Aplica pomada de vaselina oftálmica veterinário sobre os olhos para evitar ressecamento enquanto sob anestesia. Administre a anticoagulação (100 UI/kg) com fisiológico heparinizado (50 UI/mL) por injeção de veia cauda antes da cirurgia.</li>
	<li>Raspar a pele na parede abdominal anterior usando uma lâmina de barbear e limpar a pele usando solução de iodo e de solução de álcool.</li>
	<li>Realizar uma incisão laparotomia com tesoura cirúrgica e certifique-se de que a incisão é cerca de 4-5 cm de comprimento e em seguida expor a cavidade abdominal.</li>
	<li>Retraia e enrole os intestinos com gaze umedecida com soro fisiológico, preferencialmente.</li>
	<li>A aorta abdominal disse cuidadosamente.</li>
	<li>Identificar e ligar todos os pequenos ramos usando suturas de nylon monofilamento 9-0.</li>
	<li>Fixar a seção isolada (até 1 cm de comprimento) da aorta usando duas pinças vasculares. A aorta pode permanecer preso por 20-30 min.</li>
	<li>Transecto a aorta abdominal entre duas pinças usando microtesoura para criar os sites anastomótica.</li>
	<li>Lave as duas extremidades da aorta com solução salina heparinizada (50 UI/mL) para remover o sangue residual.</li>
	<li>Descole a adventícia usando microtesoura.</li>
	<li>Anastomose do enxerto com diâmetro interno de 2 mm e 1 cm de comprimento para a aorta abdominal de ratos com um padrão de sutura em forma de oito usando suturas de nylon monofilamento 9-0.</li>
	<li>Em primeiro lugar, construir quatro anastomoses de acordo com a sequência de 3, 9, 12 e 06:00 posições no lado proximal e, em seguida, se anastomosam as bordas cortadas em 4 pontos entre duas suturas. Depois de terminar a sutura proximal, sutura do lado distal pelo mesmo método. Nota: Cada ponto é necessário para garantir que o lado nativo ligeiramente é incorporado no enxerto.</li>
	<li>Remova a braçadeira distal para permitir que o sangue flua do enxerto e, em seguida, remova a braçadeira proximal.</li>
	<li>Pressione as extremidades de sutura para parar o sangramento usando uma bola de algodão estéril ou uma pequena compressa de gaze. Pressione para cerca de 3 min, a hemostasia.</li>
	<li>Retorne os intestinos na cavidade abdominal.</li>
	<li>Lave a cavidade abdominal com soro fisiológico morno com gentamicina (320 U/mL).</li>
	<li>Cosa a parede abdominal usando uma sutura de Nylon 3-0 na camada de músculo e pele, respectivamente.</li>
	<li>Coloque o rato em uma gaiola limpa e seca e coloque uma almofada de aquecimento sob a gaiola para manter a temperatura do corpo animal; Então espere o rato para se recuperar da anestesia. Cuidar do animal até que ele recuperou a consciência suficiente para manter a prostração esternal.</li>
	<li>Depois de recuperar a consciência, coloque o rato em uma única gaiola com comida e água. Aplica iodo na ferida para prevenir a infecção após a cirurgia. Retorne o rato para a companhia de outros animais até que ele se recupere totalmente.</li>
	<li>Eutanásia em ratos de acordo com as normas institucionais em pontos de tempo predeterminado.</li>
</ol>

<ol>
	<li>Coombs, K. E., Leonard, A. T., Rush, M. N., Santistevan, D. A., Hedberg-Dirk, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+effect+of+material+stiffness+on+valvular+interstitial+cell+differentiation.">Isolated effect of material stiffness on valvular interstitial cell differentiation.</a> J Biomed Mater Res A. 105 (1), 51-61 (2017).</li><li>Zhang, L., 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=A+sandwich+tubular+scaffold+derived+from+chitosan+for+blood+vessel+tissue+engineering.">A sandwich tubular scaffold derived from chitosan for blood vessel tissue engineering.</a> J Biomed Mater Res A. 77 (2), 277-284 (2006).</li><li>Thottappillil, N., Nair, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+in+vascular+regeneration:+current+status.">Scaffolds in vascular regeneration: current status.</a> Vasc Health Risk Manag. 11, 79-91 (2015).</li><li>Pektok, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+and+healing+characteristics+of+small-diameter+poly+(e-caprolactone)+vascular+grafts+in+the+rat+systemic+arterial+circulation.">Degradation and healing characteristics of small-diameter poly (e-caprolactone) vascular grafts in the rat systemic arterial circulation.</a> Circulation. 118 (24), 2563-2570 (2008).</li><li>Innocente, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paclitaxel-eluting+biodegradable+synthetic+vascular+prostheses:+a+step+towards+reduction+of+neointima+formation?.">Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?.</a> Circulation. 120 (11 Suppl), S37-S45 (2009).</li><li>de Valence, S., 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=Advantages+of+bilayered+vascular+grafts+for+surgical+applicability+and+tissue+regeneration.">Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration.</a> Acta Biomater. 8 (11), 3914-3920 (2012).</li><li>Assmann, A., 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=Acceleration+of+autologous+in+vivo+recellularization+of+decellularized+aortic+conduits+by+fibronectin+surface+coating.">Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating.</a> Biomaterials. 34 (25), 6015-6026 (2013).</li><li>Hasan, A., 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=Electrospun+scaffolds+for+tissue+engineering+of+vascular+grafts.">Electrospun scaffolds for tissue engineering of vascular grafts.</a> Acta Biomater. 10 (1), 11-25 (2014).</li><li>Baker, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29 (15), 2348-2358 (2008).</li><li>Wang, K., 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=Creation+of+macropores+in+electrospun+silk+fibroin+scaffolds+using+sacrificial+PEO-microparticles+to+enhance+cellular+infiltration.">Creation of macropores in electrospun silk fibroin scaffolds using sacrificial PEO-microparticles to enhance cellular infiltration.</a> Journal of Biomedical Materials Research Part A. 101 (12), 3474-3481 (2013).</li><li>Lee, B. L. P., 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=Femtosecond+laser+ablation+enhances+cell+infiltration+into+three-dimensional+electrospun+scaffolds.">Femtosecond laser ablation enhances cell infiltration into three-dimensional electrospun scaffolds.</a> Acta Biomaterialia. 8 (7), 2648-2658 (2012).</li><li>Rnjak-Kovacina, J., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+pore+size+of+electrospun+scaffolds.">Increasing the pore size of electrospun scaffolds.</a> Tissue Eng Part B Rev. 17 (5), 365-372 (2011).</li><li>Zhong, S., Zhang, Y., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+large+pores+in+electrospun+nanofibrous+scaffolds+for+cellular+infiltration:+a+review.">Fabrication of large pores in electrospun nanofibrous scaffolds for cellular infiltration: a review.</a> Tissue Eng Part B Rev. 18 (2), 77-87 (2012).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+poly(epsilon-caprolactone)+microfiber+and+multilayer+nanofiber/microfiber+scaffolds:+characterization+of+scaffolds+and+measurement+of+cellular+infiltration.">Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.</a> Biomacromolecules. 7 (10), 2796-2805 (2006).</li><li>Rnjak-Kovacina, 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=Tailoring+the+porosity+and+pore+size+of+electrospun+synthetic+human+elastin+scaffolds+for+dermal+tissue+engineering.">Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering.</a> Biomaterials. 32 (28), 6729-6736 (2011).</li><li>Wang, Z., 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=The+effect+of+thick+fibers+and+large+pores+of+electrospun+poly(epsilon-caprolactone)+vascular+grafts+on+macrophage+polarization+and+arterial+regeneration.">The effect of thick fibers and large pores of electrospun poly(epsilon-caprolactone) vascular grafts on macrophage polarization and arterial regeneration.</a> Biomaterials. 35 (22), 5700-5710 (2014).</li><li>Hutcheson, J. D., 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=Genesis+and+growth+of+extracellular-vesicle-derived+microcalcification+in+atherosclerotic+plaques.">Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques.</a> Nat Mater. 15 (3), 335-343 (2016).</li><li>Tara, S., 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=Well-organized+neointima+of+large-pore+poly(L-lactic+acid)+vascular+graft+coated+with+poly(L-lactic-co-epsilon-caprolactone)+prevents+calcific+deposition+compared+to+small-pore+electrospun+poly(L-lactic+acid)+graft+in+a+mouse+aortic+implantation+model.">Well-organized neointima of large-pore poly(L-lactic acid) vascular graft coated with poly(L-lactic-co-epsilon-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(L-lactic acid) graft in a mouse aortic implantation model.</a> Atherosclerosis. 237 (2), 684-691 (2014).</li></ol>

Os autores têm sem conflitos de interesses financeiros.

Infiltração de células é fundamental para a regeneração e remodelação do enxerto vascular na vivo16. Infiltração de célula limitada é muitas vezes relacionada com os poros relativamente pequenos do enxerto que dificultam a migração de células na parede da prótese. Para resolver esta dificuldade, desenvolvemos um método modificado para preparar enxertos vasculares de electrospun PCL com estrutura de poros grandes. No detalhe, o tamanho dos poros aumentou com o aumento da e...

Enxertos vasculares feitos de polímeros sintéticos são amplamente utilizados na clínica para o tratamento de doenças cardiovasculares (doenças cardiovasculares). Infelizmente, no caso de enxertos vasculares de pequeno diâmetro (D < 6mm) não há nenhuma produtos de sucesso disponíveis devido a baixa permeabilidade desencadeada pela velocidade do fluxo de sangue reduzido, que muitas vezes leva a trombose, hiperplasia da íntima e outros complicações1.
Engenharia de tecidos fornece uma estratégia alternativa para realizar a patência a longo prazo e homeostase baseado na reconstrução e regeneração vascular andaime-guiada. No detalhe, o enxerto vascular, como um modelo tridimensional, poderia fornecer suporte mecânico e orientação estrutural durante a regeneração do tecido vascular e influência funções celulares, incluindo a adesão celular, migração, proliferação, e secreção de matriz extracelular2. Até agora, vários polímeros sintéticos foram avaliados para aplicações em engenharia de tecido vascular. Entre estes polímeros, poly(ε-caprolactone) (PCL) tem sido intensamente pesquisada devido à célula boa compatibilidade e degradação lenta que variam de vários meses a dois anos3. Em um rato aorta modelo4,5,6, enxertos vasculares PCL, processados por eletrofiação exibiram excelente integridade estrutural e patência, invasão da célula, bem como continuamente aumentado e neovascularização na parede da prótese por até 6 meses. No entanto, remodela o tecido adverso, incluindo regressão de células e capilares e calcificação, também foram observados em momentos mais, acima de 18 meses.
Celularização da prótese vascular é um factor-chave determinando a regeneração do tecido e remodelando o7. Eletrofiação, como uma técnica versátil, tem sido amplamente empregada para a preparação de enxertos vasculares com estrutura fibrosa nano8. Infelizmente, a estrutura relativamente pequenos poros conduz frequentemente a infiltração de células insuficientes no enxerto vascular electrospun, que limita a regeneração do tecido subsequentes. Para resolver este problema, tem sido tentadas várias técnicas para aumentar o tamanho dos poros e porosidade total, incluindo o sal/polímero lixiviação9,10, modificação do aparelho coletor, pós-tratamento por radiação laser11 , etc. Na verdade, a estrutura dos enxertos de electrospun (incluindo o diâmetro da fibra, tamanho dos poros e porosidade) está intimamente relacionada com as condições de processamento de12,13. Durante eletrofiação, o diâmetro da fibra pode ser facilmente controlado alterando os parâmetros, tais como a concentração da solução de polímero, vazão, tensão, etc. 14 , 15, e portanto, o tamanho dos poros e porosidade foram aprimorados em conformidade.
Recentemente, informou um enxerto de electrospun PCL modificado com estrutura se (fibras com diâmetro de 5 a 7 µm e poros de 30-40 µm). Implantação in vivo , substituindo aorta abdominal de ratos mostrou alta taxa de patência, bem como a boa regeneração endotelização e músculo liso no pós-operatório de 3 meses16. Mais importante, não remodela o tecido adverso incluindo regressão calcificação e célula podia ser observada mesmo após um ano de implantação.

<table><tbody><tr><td>Poly(&amp;epsilon;-caprolactone) (PCL) pellets (M&lt;sub&gt;n&lt;/sub&gt;=80,000)</td><td>Sigma</td><td>704067</td><td></td></tr><tr><td>Methanol</td><td>Tianjin Chemical Reagent Company</td><td>1060</td><td></td></tr><tr><td>Alcohol</td><td>Tianjin Chemical Reagent Company</td><td>1083</td><td></td></tr><tr><td>Chloroform</td><td>Tianjin Chemical Reagent Company</td><td>A1007</td><td></td></tr><tr><td>Sucrose</td><td>Tianjin Fengchuan Company</td><td>2296</td><td></td></tr><tr><td>Triton X-100</td><td>Alfa Aesar</td><td>A16046</td><td></td></tr><tr><td>Sprague Dawley rats</td><td>Laboratory Animal Center of the Academy of Military Medical Sciences</td><td></td><td></td></tr><tr><td>Normal saline</td><td>Hebei Tiancheng Pharmaceutical company</td><td></td><td></td></tr><tr><td>Chloral hydrate</td><td>Tianjin Ruijinte chemical company</td><td>2223</td><td></td></tr><tr><td>Heparin sodium Injection</td><td>Tianjin Biochem Pharmaceutical company</td><td></td><td></td></tr><tr><td>Gentamycin Sulfate Injection</td><td>Jiangsu Lianshui Pharmaceutical company</td><td></td><td></td></tr><tr><td>Mouse anti-&amp;alpha;-SMA primary antibody</td><td>Abcam</td><td>ab7817</td><td></td></tr><tr><td>Mouse anti-smooth MYH primary antibody</td><td>Abcam</td><td>ab683</td><td></td></tr><tr><td>Rabbit polyclonal anti-rat elastin antibody</td><td>Abcam</td><td>ab23748</td><td></td></tr><tr><td>Rabbit anti-von Willebrand factor primary antibody</td><td>Abcam</td><td>ab6994</td><td></td></tr><tr><td>Goat anti-mouse IgG (Alexa Fluor 488)</td><td>Invitrogen</td><td>ab150117</td><td></td></tr><tr><td>Goat anti-rabbit IgG (Alexa Fluor 488)</td><td>Invitrogen</td><td>ab150077</td><td></td></tr><tr><td>5% normal goat serum</td><td>Zhongshan Golden bridge</td><td>ZLI9022</td><td></td></tr><tr><td>Hematoxylin and eosin (H&amp;amp;E)</td><td>Beijing leagene biotech</td><td>DH0006</td><td></td></tr><tr><td>Masson&#039;s trichrome</td><td>Beijing leagene biotech</td><td>DC0032</td><td></td></tr><tr><td>Verhoeff-van Gieson (VVG)</td><td>Beijing leagene biotech</td><td>DC0059</td><td></td></tr><tr><td>Von Kossa</td><td>Beijing leagene biotech</td><td>DS0003</td><td></td></tr><tr><td>Surgical sutures needles with thread,3-0 silk</td><td>Shanghai Jinhuan medical supplies company</td><td>G3002b</td><td></td></tr><tr><td>Surgical sutures needles with thread,9-0 silk</td><td>Shanghai Jinhuan medical supplies company</td><td>H901</td><td></td></tr></tbody></table>

implantation of electrospun vascular grafts with optimized structure in a rat model

Aqui, apresentamos um protocolo para fabricar se enxerto vascular de PCL e descrever um protocolo de avaliação, usando um modelo do rato de substituição da aorta abdominal. Os enxertos vasculares electrospun muitas vezes possuem relativamente pequenos poros, que limitam a infiltração de células os enxertos e dificultam a regeneração e remodelação das artérias-neo. Neste estudo, enxertos vasculares PCL com fibras mais grossas (5-6 µm) e poros maiores (~ 30 µm) foram fabricados usando uma técnica de processamento modificado. O desempenho a longo prazo do enxerto foi avaliado pela implantação de um modelo de aorta abdominal de ratos. Análise de ultrassom mostrou que os enxertos permaneceram patentes sem aneurisma ou estenose ocorrendo mesmo após 12 meses da implantação. Estrutura se melhorou o crescimento celular e assim promovido o tecido regenerado em 3 meses. Mais importante, não havia nenhum sinal de remodelação adversos, tais como calcificação na parede do enxerto após 12 meses. Portanto, enxertos vasculares de electrospun PCL com modificados se processamento têm potenciais para ser um substituto de artéria para implantação de longo prazo.

Os enxertos PCL foram explantados em 3 meses e 12 meses de pós-operatório e analisaram por técnicas histológicas padronizadas de hematoxilina e eosina (H & E), corante tricromo de Masson, Verhoeff-van Gieson (Wagner), Von Kossa e mancha para α-SMA, MYH, vWF e elastina. As imagens histológicas foram tiradas usando um microscópio na posição vertical, e as imagens de imunofluorescência foram tiradas usando um microscópio ﬂuorescence.
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Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Implantação de enxertos vasculares Electrospun com estrutura otimizada em um modelo do rato

Aqui, apresentamos um método eletrofiação modificado para fabricar enxertos vasculares PCL com fibras grossas e poros dilatados e descrever um protocolo para avaliar o desempenho na vivo em um modelo do rato de substituição da aorta abdominal.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

implantation-electrospun-vascular-grafts-with-optimized-structure-rat

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.9K visualizações.  Nankai University. Aqui, apresentamos um método eletrofiação modificado para fabricar enxertos vasculares PCL com fibras grossas e poros dilatados e descrever um protocolo para avaliar o desempenho na vivo em um modelo do rato de substituição da aorta abdominal.

Vídeo: Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Treinar um técnica microcirúrgica sofisticado: Interposição de enxerto de veia jugular externa na artéria carótida comum em ratos

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Medicina

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantação de engenharia de tecidos no coração de ratos

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Biologia

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 ...

Bioengenharia

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. ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Atímica Rat Modelo de Avaliação da Engineered Ligamento Cruzado Anterior Enxertos

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Implantação de Veia Cava Inferior interposição de enxerto no Rato Modelo

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

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 ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Enxerto projetado por tecido para reconstrução circunferencial do Esôfago em Ratos

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Veia Interposição Modelo: Um modelo adequado para estudar Bypass Graft Perviedade

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

Implantação de enxertos vasculares Electrospun com estrutura otimizada em um modelo do rato

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