The authors thank Tyra Witt, Cheri Mueske, Brant Newman and Dr. Peter J. Psaltis, MBBS, PhD for their valuable contributions. This study was financially supported by European Regional Development Fund &#8211; FNUSA-ICRC (No. CZ.1.05/1.100/02.0123), American Heart Association Scientist Development Grant (AHA #06-35185N), National Institutes of Health (T32HL007111) and The Grainger Innovation Fund &#8211; Grainger Foundation.

Todos os estudos com animais foram aprovados pelo Animal Care Institucional e Comitê de Utilização (IACUC) da Clínica Mayo. 1. Análise e Desenho de um 2205 de aço inoxidável Stent <ol><li> Projetando um nu metal stent usando CAD <ol><li> Faça um cilindro oco extrudido, selecionando em função &#39;de ressalto extrudado / base&#39; com a espessura da parede igual à espessura haste do stent. </li><li> Projetar um padrão de stent em um avião diferente esboço tangente ao cilindro extrudido. Adicione a largura do padrão plana para coincidir com a circunferência do cilindro oco extrudido. </li><li> Transferir o projeto padrão plano para o cilindro oco usando o recurso de envoltório. </li><li> Salve a peça em seu formato nativo e também em formato ACIS a ser exportado para FEA. </li></ol></li><li> Análise de elementos finitos para os modelos de stents <ol><li> Importe a geometria sólida salvo no formato ACIS no módulo de parte do software FEA para mais analysé. </li><li> Modelo 2 cilindros analíticos de forma coaxial com o stent no modelador parte do software FEA. O cilindro tem um diâmetro exterior inicial maior do que o diâmetro da prótese endovascular para simular o punção e o cilindro interior tem um diâmetro inicial de 1 mm para simular um balão de inflação. </li><li> Clique duas vezes no item da árvore &#39;casos&#39; do modelador de montagem para montar o dito acima partes em posições relativas. </li><li> Use o módulo de malha do software FEA, especifique o tipo de elemento como 20-nó de elemento de hexaedro com integração reduzida, especifique o tamanho do elemento, e malha do stent. </li><li> Especificar pares de contato rígidas sem atrito entre o stent e os dois cilindros, respectivamente, no &#39;propriedades&#39; de interação da árvore modelo. </li><li> Atribuir comportamento tensão-deformação elasto-plástica de 2205 de aço inoxidável para o modelo de stent. </li><li> Definir condições de contorno para cravar em primeiro lugar o cilindro exterior para 1 mm, o que simula o CRimping do stent. Remover o cilindro exterior para simular o relaxamento da prótese endovascular de cravamento. Expandir o cilindro interior a 3 mm para simular expansão e, finalmente, remover o cilindro interior para simular recuo da prótese endovascular. </li><li> Defina os parâmetros de simulação, incluindo o número de processadores e quantidade de RAM alocados no &#39;Análise&#39; item da árvore modelo e executar a simulação. </li><li> Uma vez que a simulação é completa, abra o ficheiro de resultado (filename.odb) e pós-processar os resultados para estudar os principais estirpes e iterativamente melhorar a concepção do stent para alcançar um principal deformação de 20%, o que é menos do que o limite de falha do material . </li></ol></li></ol> 2. Stent Fabricação e Teste de engaste e Expansão <ol><li> Fabricação Stent <ol><li> Obter os tubos de aço inoxidável 2205 por perfuração arma e precisão bar moagem material de estoque em uma empresa de usinagem de precisão, tais como produtos de precisão em Ação Pioneer,OH. </li><li> Transferir os tubos terra de precisão e design plano padrão de stent para uma empresa stent corte como LaserAge Technology Corporation em Waukegan, IL para corte a laser e eletropolimento. </li><li> Passivar a superfície dos stents eletropolidas submergindo-os em um ácido forte (50% de HCl) durante 10 min, seguido por uma base (10% de NaHCO3) durante mais 10 min. CUIDADO: lidar com produtos químicos com equipamento de proteção adequado e sob uma coifa. Finalmente, lavar os stents com álcool etílico e água desionizada. Este processo é chamado de decapagem ácida. </li></ol></li><li> Ensaios de stent fabricado para frisar e expansão <ol><li> Crimp o stent em um balão com três dobras usando uma mão ferramenta de friso. Segurar o stent eo balão com três dobras na ferramenta de compressão. Pressionar a alavanca para deformar radialmente o stent a ser cravado no balão. </li><li> Inspecione o stent frisado usando um microscópio para uniforme de cravamento e quaisquer sinais de falha na estrutura devidoà deformação plástica. </li><li> Expandi-lo para o diâmetro de 3 mm concebido pressurizando o balão com três dobras com água. Examine os stents expandidas para fraturas microscópicas e expansão uniforme. </li></ol></li></ol> 3. Caracterização de Stent para Retenção de Campo Magnético NOTA: ímã cilíndrico de 2 polegadas de diâmetro e 1 polegada de altura foi utilizado neste estudo. Os pólos do ímã estão alinhados ao longo do eixo. A densidade superficial do fluxo magnético do íman é de aproximadamente 1 T. <ol><li> Magnetizar os stents diametralmente ou axial utilizando um ímã de neodímio forte. Segurar o stent perto do forte ímã para cerca de 1 min para magnetização. </li><li> Segurar o stent em uma das faces planas com o seu diâmetro ao longo das linhas de campo magnético para ser magnetizadas ou diametralmente Segurar o stent próximo à superfície cilíndrica com o seu eixo ao longo das linhas de campo magnético para magnetizar-lo axialmente. Fiel magnético retidod do stent foi encontrado para ser estável durante pelo menos 24 horas, mas o stent usar o mais rapidamente possível depois da magnetização. </li><li> Monte os stents individualmente para mandris de vidro e, em seguida, montar os mandris de vidro no mandril de precisão do dispositivo de sondagem magnético. Sonda de micro-sensor magnético pode ser precisamente posicionada perto da prótese endovascular sem tocar na superfície utilizando o XYZ fases de montagem do dispositivo de sondagem magnética (Figura 4). </li><li> Medir a leitura da linha de base do microssensor magnética longe da prótese endovascular e, em seguida, medir o campo magnético retida na superfície da prótese endovascular através do posicionamento da sonda utilizando as fases de XYZ a fixação magnética de sondagem. </li></ol> 4. Estudos de captura celular magnéticos <ol><li> Obtenção de células, a rotulagem com spion e coloração com corante fluorescente <ol><li> Derivar as células endoteliais excrescência (EdC) do sangue periférico de porco como descrito em 5,7. A cultura num frasco T-75 until aproximadamente 80% confluentes (5x10 6 a 8x10 6 células). </li><li> Sintetizar SPIONs como a magnetita diâmetro de 10 nm (Fe 3 O 4) núcleo rodeado por 50 nm de espessura poli (ácido láctico-co-glicólico) (PLGA) shell como descrito em 8,9. </li><li> Incubar a EOC derivado com spion a uma concentração de 200 ug / ml de meio de cultura celular durante 16 horas a 37 o C. </li><li> Aspirar suavemente o meio de cultura celular. Lavar suavemente as células por adição de 10 ml de solução salina tamponada com fosfato (PBS) para o balão, de balanço, e aspirando o PBS. </li><li> Corar as células com corante fluorescente (CM-DII) para a visualização durante as experiências. Isto é feito por as instruções do fabricante, adicionando o corante para 10 ml de meio de cultura celular numa concentração de 5 ul / ml e incubou-se com as células durante 30 min a 37 ° C. </li><li> Lavam-se as células com PBS como no passo 4.1.4 e incubar com 3 ml de solução de tripsina-EDTA a 0,25% durante 5 min a 37 ° C alevantar as células do frasco. </li><li> Transferir a suspensão de células para um tubo de 15 ml, superior fora com PBS, e centrifugar a 500 xg durante 5 minutos para formar um sedimento celular. </li><li> Re-suspender o sedimento celular em PBS a uma concentração de 1-2x10 6 células / ml e misturar bem por pipetagem dentro e para fora do tubo cónico várias vezes. </li></ol></li><li> Em estudos in vitro de células- <ol><li> Concepção e fabricar (por exemplo, impressão em 3D) um acessório simples para manter o stent apenas acima da superfície de uma lamela de vidro. </li><li> Desmagnetizar um stent utilizando um desmagnetizador eletromagnética ou magnetizar um stent diametralmente ou axial utilizando um ímã de neodímio forte. </li><li> Pipeta da EOC marcado com spion suspensas em PBS no prato contendo os stents controle axialmente magnetizados ou diametralmente magnetizados ou não-magnetizados. Imagem dos stents com EOC suspensas em PBS imediatamente para a fluorescência utilizando um microscópio de fluorescência invertido. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 5. Em estudos in vivo em animais <ol><li> Implante de stent <ol><li> Extrair sangue periférico a partir de 4 porcos Yorkshire saudáveis ​​- pesando aproximadamente 50 kg - 3 semanas antes da implantação do stent, respectivamente, e a cultura EOC como descrito em 5,7. </li><li> Administrar a medicação anti-plaquetária começando 3 dias antes da cirurgia (325 mg de aspirina e clopidogrel 75 mg por dia). </li><li> No dia em que o implante de stent, anestesiar os porcos com intramuscular Telazol, xilazina e atropina (5 / 2-3 / 0,05 mg / kg, respectivamente), conforme estabelecido nos cuidados de animais e utilização diretrizes institucionais aplicáveis. </li><li> Entubar e colocar o porco em inalação de 1-2,5% anestesia isoflurano. </li><li> Raspar a região do pescoço ventral do porco e realizar o procedimento em condições estéreis gerais. </li><li> Implant 1 magnetizado e um stent não-magnetizado na artéria coronária direita (RCA) utilizando a técnica de cateterismo cardíaco normal. <ol><li> Cathetezação de animais deve ser realizado por um cardiologista intervencionista treinada. Acesse a artéria carótida direita com uma bainha 9 French. </li><li> Canular a artéria coronária alvo e injetar corante de contraste iodado para obter imagens da fluoroscopia. </li><li> Coloque um fio guia 0,014 polegadas padrão coronária na artéria. Avance o balão e stent usar este fio-guia e implantar o stent em um navio de diâmetro 3-3,5 mm. </li></ol></li><li> Ocluir o fluxo de sangue dentro do proximal RCA para os stents implantados utilizando um balão ao longo do fio e a entregar cerca de 2x10 6 EOC autólogas marcadas com spion suspenso em 4 ml de PBS através da central de cateter ao longo de um período de 2 min. </li><li> Restaurar o fluxo sanguíneo para o RCA após 2 min de oclusão adicional. </li><li> Transferir o animal para a sala de recuperação e acompanhar de perto o animal até que ele recuperou a consciência. </li><li> Continue a administrar a medicação anti-plaquetas (aspirina 325 mg e clopidogrel 75 mg) pós operatório até o sacrifício. </li></ol></li><li> Explante Stent e histologia <ol><li> Eutanásia dos animais 7 dias após a cirurgia por anestesia primeiro animal, tal como explicado anteriormente, e em seguida administrar por via intravenosa uma dose letal de pentobarbital de sódio (100 mg / kg) de acordo com cuidado e uso de animais com as orientações institucionais aplicáveis. </li><li> Cirurgicamente colher os segmentos arteriais com stent. Fixar as artérias explantados em tampão de formalina a 10% durante um período mínimo de 30 min. Deixe as amostras em tampão de formalina para análise histológica. </li><li> Terceirizar a amostra fixa às instalações capazes de realizar histologia com stents de metal. Durante este processamento, as amostras são incorporadas em metilo, em corte transversal, e analisados ​​histologicamente usando técnica de coloração de Mallory com mancha azul da Prússia para partículas de ferro. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Desenvolvemos um stent magnético que pode funcionar como um stent convencional e pode atrair células endoteliais marcado com spion. Em estudos anteriores envolvendo stents magnéticas, os pesquisadores usaram níquel stents revestidos comerciais e bobinas ou malhas feitas de materiais magnéticos devido à indisponibilidade de um stent ferromagnético 5,10-14. Outros grupos também têm utilizado a natureza paramagnética de stents de aço inoxidável 304 de grau comercialmente disponíveis para o direciona...

Patients implanted with vascular stents manufactured from thrombogenic materials like stainless steel, cobalt chromium, and platinum chromium &#8211; both bare metal stents (BMS) and drug eluting stents (DES) &#8211; need anti-platelet therapy to prevent thrombus formation. BMS heal rapidly, but are subject to late stage restenosis due to incomplete healing. DES require long term anti-platelet therapy due to delayed healing. Anti-platelet therapy administered to avoid thrombosis as a result of incomplete or delayed healing leads to increased bleeding risk and may not be suitable for certain patients1,2. An ideal stent will heal completely and quickly thus avoiding long-term anti-platelet therapy and late stage restenosis. This complete healing can only be achieved if the stent is rapidly coated with a monolayer of endothelial cells after implantation. Coating the stents with biocompatible materials such as gold or other biopolymers has been shown to improve thrombo-resistance, but none of these techniques achieved ideal blood compatibility as may be possible by coating with endothelial cells3,4.
A stent can be coated with endothelial cells post implantation by attracting circulating progenitor cells. This self-seeding technique can be achieved by utilizing ligands and antibodies. But this technique is limited by the low number of circulating endothelial progenitor cells. A promising strategy is to deliver cells directly to the stent immediately following implantation during a short period of blood flow occlusion3,5. This strategy requires a technique for rapidly capturing cells and retaining them on the stent even after restoring blood flow. We have developed a technique in which a magnetic stent is used to attract and retain magnetically-labeled endothelial cells delivered post implantation. To achieve this, a functional BMS with sufficient magnetic properties to capture and retain magnetically-labeled endothelial cells is required6.
In this paper, we discuss the methods for designing, manufacturing, and testing a 2205 stainless steel stent. The stents were designed using CAD and FEA. The manufactured stents were magnetized using a neodymium magnet and the retained magnetic field was measured using a magneto-resistance microsensor probe. We then tested the stents for magnetically-labeled cell capture in a culture dish during our in-vitro experiments. Finally, the stents were tested in-vivo by implanting magnetic and non-magnetic stents in 4 pigs and histologically analyzing the stented arteries.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2205 Stainless steel</td>
			<td>Carpenter Technology Corporation</td>
			<td>N/A</td>
			<td>Round bar stock material</td>
		</tr>
		<tr>
			<td>Abaqus</td>
			<td>Dassault systems</td>
			<td>N/A</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td></td>
			<td></td>
			<td>Prescription drug.</td>
		</tr>
		<tr>
			<td>Clopidogrel</td>
			<td></td>
			<td></td>
			<td>Commercial name: Plavix. Prescription drug.</td>
		</tr>
		<tr>
			<td>CM-DiI</td>
			<td>Life Technologies</td>
			<td>V-22888</td>
			<td>Molecular Probes, Eugene, OR</td>
		</tr>
		<tr>
			<td>Endothelial growth medium-2</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand Held Crimping tool</td>
			<td>Blockwise engineering</td>
			<td>M1-RMC</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00011324</td>
			<td>CAUTION: wear proptective equipment and handle under fume hood</td>
		</tr>
		<tr>
			<td>Isoflurane anesthesia</td>
			<td>Piramal Critical Care, Inc.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00011674</td>
			<td></td>
		</tr>
		<tr>
			<td>NdFeB magnet 2&#34; Dia x 1&#34; thick</td>
			<td>Amazing magnets</td>
			<td>D1000P</td>
			<td>Axially magnetized disc magnet with poles on flat faces</td>
		</tr>
		<tr>
			<td>Over-The-Wire trifold balloon</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any commercially available OTW trifold balloon can be used</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td>Commonly known as PBS</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00003528</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Zoetis</td>
			<td></td>
			<td>Commercial Name: Sleepaway (26%), FatalPlus, Beuthanasi.&#160; Controlled substance to be ordered only by licensed veternarian</td>
		</tr>
		<tr>
			<td>SolidWorks</td>
			<td>Dassault systems</td>
			<td>N/A</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>SpinTJ-020 micro sensor</td>
			<td>MicroMagneitcs Sensible Solutions</td>
			<td>N/A</td>
			<td>Long probe STJ-020 microsensor</td>
		</tr>
		<tr>
			<td>SPION</td>
			<td>Mayo Clinic</td>
			<td>N/A</td>
			<td>Nanoparticles synthesized internally (Ref: Lee, S. J. et al. Nanoparticles of magnetic ferric oxides encapsulated with poly(D,L latide-co-glycolide) and their applications to magnetic resonance imaging contrast agent. J Magn Magn Mater 272, 2432-2433, doi:DOI 10.1016/j.jmmm.2003.12.416 (2004))</td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td></td>
			<td>Controlled substance to be ordered only by licensed veternarian</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td>Gibco, Grand Island, NY</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer Animal Health</td>
			<td></td>
			<td>Commercial name: Rompun. Controlled sunstance to be ordered only by a licensed veternarian</td>
		</tr>
	</tbody>
</table>

ferromagnetic bare metal stent for endothelial cell capture and retention

Endotelização rápida de stents cardiovasculares é necessária para reduzir a trombose de stent e para evitar a terapia anti-plaquetas que podem reduzir o risco de sangramento. A viabilidade do uso de forças magnéticas para capturar e reter as células endoteliais excrescência (EdC) marcadas com nanopartículas de óxido de ferro super-paramagnéticas (spion) foi mostrado anteriormente. Mas esta técnica requer o desenvolvimento de um stent funcional mecanicamente a partir de um material magnético e biocompatível seguido de in-vitro e in-vivo de ensaio para comprovar rápida endotelização. Nós desenvolvemos um stent fracamente ferromagnéticos de aço inoxidável 2205 duplex utilizando desenho assistido por computador (CAD) e seu design foi aperfeiçoada por meio de análise de elementos finitos (FEA). O desenho final do stent exibiu a principal pressão abaixo do limite de fratura do material durante cravamento mecânica e expansão. Cem stents foram fabricados e um subconjunto deles foi usado para ensaios mecânicos, tempo de retained medições do campo magnético, em estudos in vitro de captura de células, in vivo e estudos de implantação. Dez stents foram testados para implantação para verificar se eles sustentado friso e expansão ciclo sem falhas. Outros 10 stents foram magnetizadas usando um ímã de neodímio forte e seu campo magnético retido foi medido. Os stents mostrou que o magnetismo retido foi suficiente para capturar spion marcado EOC nos nossos estudos in vitro. Foi verificada marcado com spion captura e retenção de EOC em grandes modelos animais através da implantação de um stent magnetizado e um stent controle não-magnetizado em cada um dos 4 porcos. As artérias stented foram explantadas após 7 dias e analisados ​​histologicamente. Os stents fracamente magnéticos desenvolvidas neste estudo foram capazes de atrair e reter as células endoteliais marcado com spion que podem promover a cura rápida.

<html> Desenho de stent iterativo com base em FEA (Figura 1) revelou um stent que pode friso e expandir-se com uma entidade de deformação de 20%, o que é menos do que a estirpe final de 30%. Engaste e ensaio de expansão (Figura 2) não mostrou sinais de fractura. Fotos do stent deformado mostrou boa concordância com deformações FEA calculados e também imagens de microscopia mostrou nenhuma fratura (Figura 3). Como esperado a partir das medições do campo magnét...

Watch this Scientific Journal Video about Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention at JoVE.com

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Nossos objetivos foram para projetar, fabricar e testar stents ferromagnéticos para a captura de células endoteliais. Dez stents foram testados quanto à fractura e mais 10 stents foram testados para o magnetismo retida. Finalmente, 10 stents foram testados in vitro e 8 mais stents foram implantados em 4 porcos para mostrar a captura e retenção celular.

Ferromagnético Bare Metal Stent para captura de células endoteliais e Retenção

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

ferromagnetic-bare-metal-stent-for-endothelial-cell-capture

Research

JoVE Journal

Bioengineering

11.4K visualizações.  Mayo Clinic.  Nossos objetivos foram para projetar, fabricar e testar stents ferromagnéticos para a captura de células endoteliais. Dez stents foram testados quanto à fractura e mais 10 stents foram testados para o magnetismo retida. Finalmente, 10 stents foram testados in vitro e 8 mais stents foram implantados em 4 porcos para mostrar a captura e retenção celular. 

Vídeo: Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autólogas progenitoras endoteliais Cell Technology Semear e Teste de biocompatibilidade para dispositivos Cardiovascular em Modelo Animal Grande

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Bioengenharia

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Monitorização da Mecânica de parede durante a implantação de stent em um vaso

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Elétrica Cell-substrato impedância Sensing para a quantificação do endotelial Proliferação, Barreira Function, e Motilidade

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabricação de Mecanicamente Tunable e Bioativos metal andaimes para aplicações biomédicas

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

Co transplante de tecido ovariano humano com células endoteliais engenharia: uma combinação de estratégia baseada em célula acelerado perfusão com entrega direta parácrina

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Imobilização de indivíduos ao vivo Caenorhabditis elegans , usando um Chip de Microfluidic polidimetilsiloxano ultrafino com retenção de água

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Crescimento de Camadas de Células Endoteliais De Córnea Humana e Ovelha em Membranas Biomateriais

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

Mitigação do apego de células transmitidas sanguíneas a implantes metálicos através da imobilização de peptídeos derivados do CD47

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentando o crescimento de células endoteliais em placas de 6-bem em um agitador orbital para estudos mecanobiológicos

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Um sistema microfisiológico para estudar a interação das células leucócitos-endoteliais durante a inflamação

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...