Este trabalho foi apoiado pelo Departamento de Engenharia Biomédica da Universidade Case Western Reserve, tanto através laboratório start-up fundos (J. Capadona) ea Medtronic graduação Fellowship (K. Potter). O financiamento adicional sobre esta pesquisa foi apoiada em parte pela NSF concessão ECS-0621984 (C. Zorman), o caso Alumni Association (C. Zorman), o Departamento de Assuntos de Veteranos através de uma revisão Prêmio Mérito (B7122R), bem como o Advanced Plataforma Tecnológica Center (C3819C).

1. Preparação da Amostra <ol><li> Prepare a película de PVAc-NC de espessura na gama de 25-100 mM, utilizando uma técnica de moldagem em solução e compressão 10-12. </li><li> Aderem filme a uma pastilha de silício, por aquecimento sobre uma placa quente durante dois minutos, a 70 ° C (acima da temperatura de transição de vidro) a promover um contacto íntimo entre a película e a bolacha. Este passo assegura que a película preparada permanece estável e fixo para a bolacha de Si, o que é necessário para os processos microtorneamento planares. </li><li> Padrão do filme na amostra geometrias teste de-micro-usinagem a laser (VLS 3,50, VersaLASER). Defina os dois parâmetros de micro-usinagem a laser direto escrever CO para 1,0% de energia (0,5 W), a velocidade de 4,0% (56 mm / s), e 1000 pulsos por polegada 13,16. </li><li> Amostras padrão que serão utilizados para estabelecer as condições ambientais (&quot;amostras de configuração&quot;) em estruturas dogbone em forma de almofada com as dimensões laterais de 1,5 x 1,5 mm 2, e feixe lateral, dimensões 300 x 3000 mM 2, com uma espessura correspondente que ao longo do filme (Figura 2). </li><li> Padrão das amostras para as experiências ex vivo (&quot;Amostra de implante&quot;) em vigas 300 mm x 6 mm, com uma espessura correspondente ao do filme. </li><li> Libertar cuidadosamente as amostras a partir da bolacha usando uma lâmina de barbear e uma pinça. </li><li> Para o manuseio da amostra, preparar os titulares de acrílico personalizado usinados destinados a servir como parte do sistema de aderência no testador de microtração. Marcações gravadas a laser mostram a linha central do suporte e 1,5 mm a partir da extremidade. Colocar uma pequena quantidade de adesivo à base de gel de cianoacrilato sobre a linha central do suporte de acrílico e aderir cuidadosamente mm de comprimento da amostra 1,5 implante ao detentor e sobrepor-se à linha de centro (fig. 3). Cada amostra de implante requer um suporte de acrílico. Tenha o cuidado de garantir que o gel adesivo permanece apenas ao longo do comprimento mm de PVAc-NC 1.5 sendo respeitado o acrtitular ylic. Caso contrário, o gel adesivo podem interferir com o comportamento mecânico da amostra. </li><li> Remover humidade de todas as amostras colocando-as num exsicador durante pelo menos 24 horas. </li><li> Medir o comprimento, largura, espessura e as dimensões das amostras usando um microscópio óptico. </li></ol> 2. Estabelecer Condições Ambientais <ol><li> Carregar um extracto seco de configuração para o testador microtração (ver Figura 4), ​​o primeiro aperto entre as garras móveis, em seguida, entre as garras fixas. </li><li> Montar uma escova de ar, com um reservatório cheio de água, para uma posição fixa, com o bocal voltado para a amostra microtração. Ligue a escova de ar de um compressor de ar através de tubos de plástico. Com o bocal de escova de ar completamente fechada, ligar o compressor de ar. </li><li> Comece procedimento de teste de microtração cíclico, alternando entre a tensão de tração (tensão positiva) e tensão de compressão (pressão negativa) aplicada a tele amostra, mantendo-se dentro da região elástica linear da trama de tensão-deformação. Para PVAc-NC, a estirpe aplicada é limitada a menos do que 2%. Nas testador microtração costume utilizado nestas experiências, a velocidade de deformação foi controlada enquanto que a força necessária para conseguir essa estirpe foi medido. Alternativamente, uma configuração diferente pode envolver o controlo da força aplicada durante a medição da tensão resultante. </li><li> Aumentar gradualmente o fluxo a partir do bocal escova de ar, e monitorizar o declive do gráfico de tensão-deformação em função da quantidade de fluxo a partir da escova de ar. O fluxo máximo que não cause uma redução significativa (&gt; 10%) no módulo de elasticidade ao longo de um período de 60 segundos é o nível que será utilizada para as experiências ex vivo. Neste ponto, as condições de humidade que não seja molhada de uma amostra seca (e, assim, contribuir para uma redução no módulo de Young) e também minimizar a amostra de secagem depois de ser exposto aos fluidos biológicos in vivo têm sido ESTAcida. </li><li> Medir a temperatura próxima da amostra. Uma configuração ideal seria incluir um termopar com leitura digital, e ser executada enquanto o aerógrafo está operando. Definir a intensidade e da distância da fonte de calor radiante de tal modo que a temperatura da amostra é mantida a 37 ° C, para se adequar às condições fisiológicas. </li></ol> 3. Compare Controle Ambiental para Controle de Não-Ambiental <ol><li> As amostras adicionais para Imergir pelo menos 30 min em solução salina tamponada com fosfato. Após este período de tempo, a amostra é completamente saturada e foi reduzido para o seu valor mínimo do módulo de Young, a uma dada temperatura. </li><li> Carregar rapidamente uma amostra para o testador de microtração e começar os testes de microtração cíclica, com o air brush off, enquanto seca a amostra. Isso vai determinar a rapidez com que a secagem da amostra em condições não controladas. </li><li> Coloque uma segunda amostra de configuração PBS-saturada no testador de microtração, e começar os testes de microtração cíclicacom a escova de ar ligado. Isso vai determinar a rapidez com que a seca de amostra em condições ambientais controladas. </li></ol> 4. Implantação da sonda e explantes <ol><li> Anexar amostra implante a um micromanipulador braçadeira e da posição ortogonal para o tecido cortical. </li><li> Antes da inserção, manter o tecido suficientemente úmido com solução salina para garantir a homogeneidade da mecânica de tecido. </li><li> Diminuir a amostra de polímero para o córtex utilizando os comandos manuais micromaniplador. Adicione amostra no tecido cortical até que o tempo de implante de destino, em geral, entre 1 e 30 min. Para evitar que o tecido de secagem para os momentos mais 5 min, levemente dab o tecido a cada 5 minutos com um cotonete embebido em soro fisiológico. </li><li> Enquanto a sonda estiver implantada no córtex, preparar o aparelho de teste de microtração para o carregamento da amostra actualmente implantado, definindo a haste de condução para a posição zero de deslocamento de 3,0 mm a partir da braçadeira amostra estacionária. Além disso, defina o bocal de escova de arpara o ajuste do fluxo e da fonte de calor radiante para determinar a intensidade adequada no passo 2.4. </li><li> No final do tempo de implante especificado, elevar a sonda do córtex utilizando os comandos manuais micromaniplador. Imediatamente, e cuidadosamente, remover a amostra da braçadeira micromanipulador e carregar para o testador microtração, tal como descrito em mais pormenor no passo 5.2. </li></ol> 5. Teste de microtração de amostras de implantes <ol><li> Para poupar tempo, após o explante, assegurar que o testador microtração é completamente pronto para aceitar a amostra de implante antes da implantação, tal como descrito na etapa 4.4. </li><li> Imediatamente após explante, coloque a amostra entre os dois conjuntos de grampos testador microtração. Uma vez que a amostra é montada num suporte de acrílico concebido para servir como a metade superior de uma pinça, o lugar de montagem da amostra sobre o implante o aperto, do lado da amostra celular baixo. É importante assegurar que a amostra é montada de tal modo que a tensão é aplied apenas ao longo do comprimento da sonda a fim de evitar a aplicação de binário à amostra durante o teste. Como tal, a amostra tem de ser montado ao centro de cada grampo, e as pinças devem ser nivelada em relação uns aos outros. </li><li> Ajustar a posição da amostra de tal modo que a distância entre as pinças é 3,0 mm, e a extremidade da sonda é colocada no grampo fixo. Este comprimento de 3,0 milímetros entre as braçadeiras é o comprimento de medida para a amostra, e será utilizado nos cálculos posteriores para determinar a pressão sobre a amostra. </li><li> Imediatamente após a obtenção da amostra entre as duas pinças, e dentro de 2 min de explante a partir do tecido neural, activar o motor na direcção de tracção para alongar a amostra a uma taxa constante (10 um / s usados ​​aqui), enquanto que, simultaneamente, a medição e registo alongamento da amostra (através de um indicador de deslocamento, Mitutoyu 543-561) e força associada (usando uma célula de carga, Técnicas de Transdutores MDB-2.5) necessária para esticar a amostra. </li><li&gt; Deter microtração em caso de falha mecânica da amostra, ou quando o fim do intervalo de varão de accionamento é alcançado. Exportar os dados coletados, e realizar a análise de dados. </li><li> Repetir o teste de microtração para cada amostra e / ou cada conjunto de condições (ou seja, tempo de inserção). </li></ol> 6. Análise de Dados <ol><li> Converter os dados brutos de alongamento a uma tensão de engenharia aplicada ao implante amostra dividindo a distância de alongamento pelo comprimento do calibre inicial, como descrito na Equação 1, onde a estirpe está ε aplicada, t é o tempo, d é o deslocamento medido pelo micrómetro indicador, e L 0 é o comprimento inicial de medida da amostra: <img src="/files/ftp_upload/50078/50078eq1.jpg" alt="Equação 1" fo:src="/files/ftp_upload/50078/50078eq1highres.jpg"/> (1) </li><li> Converter os dados de força bruta para a tensão de projecto na amostra dividindo a força (em Newtons), por traárea da secção transversal nsverse, como descrito na Equação 2: <img src="/files/ftp_upload/50078/50078eq2.jpg" alt="Equação 2" fo:src="/files/ftp_upload/50078/50078eq2highres.jpg"/> (2) onde σ é a tensão sobre a amostra, F é a força medida pela célula de carga (em Newtons), 0 w é a largura inicial da amostra, e t é 0 a espessura inicial da amostra. </li><li> Traçar a tensão (σ [t]) versus tensão (ε [t]) curva para cada amostra, utilizando um programa de computador, como o Microsoft Excel. </li><li> Isolar a parte elástica linear da trama e usar as ferramentas de ajuste de curva baseada em software para encontrar a melhor linha de ajuste a esta parte. A inclinação da linha de melhor ajustamento corresponde ao módulo de elasticidade da amostra. A porção isolada da trama deve incluir pelo menos 10 pontos de tensão-deformação, e deve ser feita a partir da porção da trama, onde a inclinação é maior. </li><li> Para os testes cíclicos, o módulo de Young vai precisar de ser determinado para cada ciclo. Isto pode ser realizado manualmente ou automatizados. </li><li> Para todos os ensaios cíclicos, plot o módulo de Young de cada ciclo em relação ao tempo. Isso indica como a variação do módulo de medição com o tempo, o que é indicativo de quão rapidamente uma amostra de configuração é molhar ou secagem. </li><li> Para amostras de implante, cada uma das amostras e o tempo de implante corresponde a um único ciclo de testes cíclicos. Medir o módulo de Young, utilizando o procedimento descrito acima para cada uma das amostras do implante. </li><li> Traçar o módulo de Young do implante em função do tempo. Neste ponto, podem ser feitas comparações com as investigações de bancada, etc </li></ol>

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O avanço dos sistemas microeletromecânicos biomédicos implantáveis ​​(bioMEMS) para interagir com os sistemas biológicos está motivando o desenvolvimento de novos materiais com propriedades altamente personalizadas. Alguns destes materiais são desenhados para apresentar uma mudança nas propriedades do material, em resposta a um estímulo encontrado no ambiente fisiológico. Uma classe, recentemente desenvolvida de materiais responde à presença de hidrogénio líquidos de formação de ligações (por e...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University Wafer</td>
			<td>&nbsp;</td>
			<td>Mechanical grade</td>
		</tr>
		<tr>
			<td>Extruded acrylic sheet</td>
			<td>Professional Plastics</td>
			<td>SACR 062EF</td>
			<td>Thickness 0.062"</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>McMaster-Carr</td>
			<td>3962A3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>McMaster-Carr</td>
			<td>8384A47</td>
			<td>#5 tip</td>
		</tr>
		<tr>
			<td>Super Glue Gel</td>
			<td>Loctite</td>
			<td>130380</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Air Brush</td>
			<td>Snap-on Industrial</td>
			<td>BF175TA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Air Compressor</td>
			<td>Paasche</td>
			<td>B002YKN8YO</td>
			<td>D500</td>
		</tr>
		<tr>
			<td>Thermocouple</td>
			<td>Omega</td>
			<td>HH12A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Cimarec</td>
			<td>SP131325Q</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CO2 direct-write laser</td>
			<td>VersaLaser</td>
			<td>3.5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dessicator</td>
			<td>Fisher Scientific</td>
			<td>08-595</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lamp</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>custom-built</td>
		</tr>
		<tr>
			<td>Microtensile tester</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>custom-built</td>
		</tr>
	</tbody>
</table>

environmentally-controlled microtensile testing of mechanically-adaptive polymer nanocomposites for ex vivo characterization

Microdispositivos implantáveis ​​estão ganhando atenção significativa para diversas aplicações biomédicas 1-4. Tais dispositivos têm sido feitas a partir de uma variedade de materiais, cada um deles oferecendo as suas próprias vantagens e deficiências 5,6. Mais importante ainda, devido às dimensões do dispositivo de microescala, é exigido um elevado módulo de elasticidade para facilitar o implante em tecidos vivos. Por outro lado, a rigidez do dispositivo deve coincidir com o tecido circundante para minimizar a tensão induzida locais 7-9. Por isso, desenvolveu recentemente uma nova classe de materiais bio-inspirados para atender a esses requisitos, respondendo aos estímulos do ambiente, com uma mudança nas propriedades mecânicas 10-14. Especificamente, o nosso nanocompósito com base em (acetato de vinilo) poli (PVAc-NC) mostra uma redução na rigidez, quando expostos à água e temperaturas elevadas (por exemplo, temperatura corporal). Infelizmente, existem alguns métodos para quantificar a rigidez dos materiais in vivo 15 e mecanismotestes mecânicos fora do ambiente fisiológico requer frequentemente grandes amostras inadequado para a implantação. Além disso, os materiais de estímulos-resposta pode recuperar rapidamente a sua rigidez inicial após remoção. Por conseguinte, temos desenvolvido um método através do qual as propriedades mecânicas de microamostras implantadas pode ser medida ex vivo, com as condições fisiológicas simuladas mantida utilizando controlo de temperatura e humidade 13,16,17. Para este efeito, um aparelho de teste de microtração costume foi concebido para acomodar amostras microescala 13,17 com muito variados módulos de Young (intervalo de 10 MPa a 5 GPa). Como os nossos interesses estão no pedido de PVAc-NC como substrato sonda neural biologicamente adaptável, uma ferramenta mecânica capaz de caracterização de amostras em microescala era necessário. Esta ferramenta foi adaptado para fornecer humidade e controlo da temperatura, o que minimiza a secagem e arrefecimento da amostra 17. Como resultado, o mecânicocaracterísticas da amostra al explantada refletir atentamente aqueles da amostra pouco antes de explante. O objetivo geral deste método é avaliar quantitativamente a nas propriedades mecânicas in vivo, especificamente o módulo de Young, de matérias estímulos-resposta, mecanicamente adaptativo polímero à base. Isto é conseguido através do estabelecimento de primeiro as condições ambientais que minimizem uma mudança na amostra propriedades mecânicas após o explante, sem contribuir para a redução da rigidez independente da que resulta da implantação. As amostras são, então, preparado para o implante, o manuseamento, e ensaio (Figura 1A). Cada amostra é implantado no córtex cerebral de ratos, que está aqui representado como um cérebro de rato explantado, por um período especificado (Figura 1B). Neste ponto, a amostra é explantado e imediatamente carregadas no testador microtração, e, em seguida, submetido a ensaios de tracção (Figura1C). Posterior análise de dados fornece insights sobre o comportamento mecânico destes materiais inovadores no ambiente do córtex cerebral.

As propriedades mecânicas de quase todos os materiais poliméricos, incluindo o nosso PVAc-CN, estão dependentes da exposição a condições ambientais. Mais notavelmente, que incluem a exposição ao calor e à umidade. Quando um material é plastificado, devido à absorção de umidade, ou sofre uma transição térmica, apresenta uma redução no módulo de Young. Na preparação da humidade ambiente e temperatura controlada por ex vivo da amostra caracterização mecânica, é importante para assegurar q...

Watch this Scientific Journal Video about Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization at JoVE.com

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Um método pelo qual é discutida a<em&gt; In vivo</em&gt; Comportamento mecânico dos materiais que respondem a estímulos é monitorizada como uma função do tempo. As amostras são testadas<em&gt; Ex vivo</em&gt; Utilizando um aparelho de microtração com controles ambientais para simular o ambiente fisiológico. Este trabalho promove ainda mais a compreensão do<em&gt; In vivo</em&gt; Comportamento do nosso material.

Teste de microtração ambientalmente controlado mecanicamente adaptativas nanocompósitos poliméricos para<em&gt; Ex vivo</em&gt; Caracterização

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

environmentally-controlled-microtensile-testing-mechanically-adaptive

Research

JoVE Journal

Bioengineering

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

10.1K visualizações.  Louis Stokes Cleveland Department of Veterans Affairs Medical Center. Um método pelo qual é discutida a In vivo Comportamento mecânico dos materiais que respondem a estímulos é monitorizada como uma função do tempo. As amostras são testadas Ex vivo Utilizando um aparelho de microtração com controles ambientais para simular o ambiente fisiológico. Este trabalho promove ainda mais a compreensão do In vivo Comportamento do nosso material.

Vídeo: Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Bioengenharia

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Ex vivo Mimetismo de normal e anormal Hematopoese Humanos

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinação da taxa de transporte de xenobióticos e nanomateriais através da placenta usando o<em&gt; Ex vivo</em&gt; Modelo de perfusão da placenta humana

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imagiologia desnaturadas colágeno Vertentes<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via foto desencadeada Hibridização do Caged Colágeno mimético Peptídeos

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Caracterização biomecânica de tecidos moles Humanos Usando recuo e testes de tração

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Um novo modelo Ex Vivo para a avaliação de desempenho de Material de injeção submucosa endoscópica

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

Métodos para testes biomecânicos in vivo em plexo braquial em leitões neonatais

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been fabricated with hydroxyapatite to facilitate cell adherence, proliferation, and osteogenesis. In this study, hybrid nanocomposites were successfully developed using graphene nanoribbons (GNRs) and nanoparticles of hydroxyapatite (nHAPs), that when employed in bioactive scaffolds may potentially improve bone tissue regeneration. These nanostructures can be biocompatible. Here, two approaches were used for preparing the novel materials. In one approach, a co-functionalization strategy was used where nHAP was synthesized and conjugated to GNRs simultaneously, resulting in nanohybrids of nHAP on GNR surfaces (denoted as nHAP/GNR). High-resolution transmission electron microscopy (HRTEM) confirmed that the nHAP/GNR composite is comprised of slender, thin structures of GNRs (maximum length of 1.8 &#181;m) with discrete patches (150-250 nm) of needle-like nHAP (40-50 nm in length). In the other approach, commercially available nHAP was conjugated with GNRs forming GNR-coated nHAP (denoted as GNR/nHAP) (i.e., with an opposite orientation relative to the nHAP/GNR nanohybrid). The nanohybrid formed using the latter method exhibited nHAP nanospheres with a diameter ranging from 50 nm to 70 nm covered with a network of GNRs on the surface. Energy dispersive spectra, elemental mapping, and Fourier transform infrared (FTIR) spectra confirmed the successful integration of nHAP and GNRs in both nanohybrids. Thermogravimetric analysis (TGA) indicated that the loss at elevated heating temperatures due to the presence of GNRs was 0.5% and 0.98% for GNR/nHAP and nHAP/GNR, respectively. The nHAP-GNR nanohybrids with opposite orientations represent significant materials for use in bioactive scaffolds to potentially promote cellular functions for improving bone tissue engineering applications.

Novel nanocomposites of graphene nanoribbons and hydroxyapatite nanoparticles were prepared using solution-phase synthesis. These hybrids when employed in bioactive scaffolds can exhibit potential applications in tissue engineering and bone regeneration.

Síntese de Nanocompositos de Grafeno-Hidroxiapatita para uso potencial em engenharia de tecido ósseo

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been ...

Estratégias de controle e monitoramento microbiano para ambientes de salas limpas

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP&lt;71&gt;) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

Autor em destaque: Estratégias de controle e monitoramento microbiano para ambientes de sala limpa e terapias celulares  

The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards.

Estratégias de Controle e Monitoramento Microbiano para Ambientes de Salas Limpas e Terapias Celulares

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical ...