Os seguintes protocolos são aceitos sob cuidado de Animal institucional da Universidade de Maryland e Comissão de utilização, bem como o Conselho de revisão institucional. Os protocolos seguem normas federais, estaduais e locais, e as diretrizes estabelecem pela política de Universidade de Maryland em biossegurança.
1. dissecação da amostra de olho
<ol><li>
Obter uma amostra de olho do banco local de matadouro ou tecido. Se um globo de olho inteiro é obtido, extrair-se imediatamente a lente, zónulas anexadas e vítreo. Nota: Os detalhes específicos descritos a seguir referem-se aos olhos dos suínos e humanos.<ol>
<li>Usando a tesoura cirúrgica desinfectada e fórceps, cortar e remover todo o tecido em excesso em torno da esclera.</li>
		<li>Firmemente manter o olho em seu lado e, usando uma lâmina de barbear, faça uma pequena incisão ao longo do lado do olho 3 mm longe da córnea. Fazer a corte profunda o suficiente para ter atingido o vítreo dentro do olho.</li>
		<li>Com uma tesoura, corte com cuidado mais longo a incisão ao redor da circunferência do olho. Evite perfurar a lente. Uma imagem representativa é mostrada na Figura 2.</li>
		<li>Uma vez que a circunferência externa do olho foi cortada, remova o tecido posterior do olho usando fórceps. Isole a lente, zónulas, corpo ciliar e o vítreo anexado com fórceps. Uma imagem representativa é mostrada na Figura 2B.</li>
		<li>Usando a tesoura e pinça, remova o vítreo em excesso para que a lente pode deitado na MLS. Nota: Em casos de transplante de córnea, o botão corneano é usado na cirurgia e o restante do globo está disponível para fins de investigação. No entanto, este globo parcial ainda pode ser usado na preparação da instalação do esticador de lente tecido. Se apenas a parte posterior é obtida, apenas execute o passo 1.1.4–1.1.5.</li>
	</ol>
</li>
	<li>Desinfecte todos pós-dissecção de equipamentos usados em solução de água sanitária de 15% por 30 min.</li>
</ol>2. julgamento montagem da lente Manual maca
<ol><li>Insira as solas de sapato de 10 mm e os topos de sapato correspondente na placa inferior da MLS então ainda há uma lacuna de 5 mm entre a parede traseira do travessão, sapato e o sapato em si.</li>
	<li>Alinhar as placas superior e inferior, encaixe as placas juntos; o dispositivo está agora na posição unstretched.</li>
	<li>Insira as placas no caso a placa e o parafuso de bloqueio no orifício localizado na parte lateral da placa de fundo.</li>
	<li>Insira o caso da placa na base e coloque a chave para os travessões alinhados.</li>
	<li>Gire a chave no sentido horário até atingir o parafuso de parada para os sapatos do contrato e torcer volta anti-horário para devolver os sapatos para a posição original unstretched.</li>
</ol>3. montagem da lente
<ol><li>Insira as solas de sapato de 10 mm na placa inferior na MLS para que um 5mm gap entre restos a parede traseira do travessão o sapato e o sapato em si.</li>
	<li>Usando pinças curvas, coloque o lente extraído virado para cima no meio da placa de fundo para que os sapatos estão apoiando a lente sobre o furo central.</li>
	<li>Encaixe o topo correspondente dos sapatos no lugar, recorte apenas as zónulas e o vítreo. Visualmente, assegurar que a lente se mantém como centrado possível na placa inferior.</li>
	<li>Repita as etapas 2.3 – 2.4.</li>
</ol>4. medição da lente
<ol><li>Coloca um sistema de imagem diretamente acima do aparelho para capturar vídeos e imagens do processo de alongamento. Não se esqueça de incluir uma régua na moldura do quadro com precisão imagens de tamanho e escala no pós-processamento. Nota: Qualquer sistema de imagem adequado é suficiente para esta etapa; Aqui usamos um 12 megapixel, focagem automática smartphone um pé da amostra.</li>
	<li>Com firmeza, mas suavemente, gire a chave no sentido horário para esticar a lente. A Figura 3 mostra imagens representativas no estado unstretched e esticada.</li>
	<li>Depois de fotografar a lente esticada, gire a chave no sentido anti-horário para restaurar a amostra em seu estado de repouso. Nota: É imperativo que a medida da lente é realizada em tempo hábil para minimizar a desidratação da lente.</li>
	<li>Fotografe claramente o estado de repouso final da lente.</li>
</ol>5. análise de dados
<ol><li>Fazer o upload da imagem para ImageJ e, use o recurso de "ponto" para selecionar pelo menos 40 pontos em torno da circunferência da lente conforme mostrado na Figura 4. Use a opção de "Medir" → "Analisar" para produzir a localização de cada ponto selecionado.</li>
	<li>Pontos de ajuste (usando o software , por exemplo, MATLAB) os local a fim de produzir um raio e qui-quadrado do ajuste conforme mostrado na Figura 4B. Converta o pixel radius e erro em métricas usando a régua fotografada.</li>
	<li>Execute duas caudas teste t pareado para comparar uma lente individual antes e depois alongamento da MLS.</li>
</ol>

<ol>
	<li>Von Helmholtz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uber+die+akkommodation+des+auges.">Uber die akkommodation des auges.</a> Arch Ophthal. 1, 1-74 (1855).</li><li>Schachar, R. A., Black, T. D., Kash, R. L., Cudmore, D. P., Schanzlin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+accommodation+and+presbyopia+in+the+primate.">The mechanism of accommodation and presbyopia in the primate.</a> Ann Ophthalmol. 27, 58-67 (1995).</li><li>Glasser, A., Campbell, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presbyopia+and+the+optical+changes+in+the+human+crystalline+lens+with+age.">Presbyopia and the optical changes in the human crystalline lens with age.</a> Vision Res. 38 (2), 209-229 (1998).</li><li>Reilly, M. A., Hamilton, P. D., Perry, G., Ravi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+behavior+and+natural+and+refilled+porcine+lenses+in+a+robotic+lens+stretcher.">Comparison of the behavior and natural and refilled porcine lenses in a robotic lens stretcher.</a> Exp Eye Res. 88, 483-494 (2009).</li><li>Langenbucher, A., Huber, S., Nguyen, N. X., Seitz, B., Gusek-Schneider, G. C., K&#252;chle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+accommodation+after+implantation+of+an+accommodating+posterior+chamber+intraocular+lens.">Measurement of accommodation after implantation of an accommodating posterior chamber intraocular lens.</a> J Cataract Refract Surg. 29 (4), 677-685 (2003).</li><li>Ehrmann, K., Ho, A., Parel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+analysis+of+the+accommodative+apparatus+in+primates.">Biomechanical analysis of the accommodative apparatus in primates.</a> Clin Exp Optom. 91 (4), 411 (2008).</li><li>Pinilla Cort&#233;s, 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=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926 (2015).</li><li>Fisher, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elastic+constants+of+the+human+lens.">The elastic constants of the human lens.</a> J Physiol. 212 (1), 147-180 (1971).</li><li>Eppig, T., 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=Biomechanical+eye+model+and+measurement+setup+for+investigating+accommodating+intraocular+lenses.">Biomechanical eye model and measurement setup for investigating accommodating intraocular lenses.</a> Z Med Ohys. 23 (2), 144-152 (2013).</li><li>Manns, F., Parel, , 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=Response+of+Human+and+Monkey+Lenses+in+a+Lens+Stretcher.">Response of Human and Monkey Lenses in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 48 (7), 3260 (2007).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+age-related+stiffening+in+the+crystalline+lens+by+Brillouin+optical+microscopy.">In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy.</a> Biophys J. 101 (6), 1539-1545 (2011).</li><li>Besner, S., Scarcelli, G., Pineda, R., Yun, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Brillouin+Analysis+of+the+Aging+Crystalline+Lens.">In Vivo Brillouin Analysis of the Aging Crystalline Lens.</a> Invest Ophthalmol Vis Sci. 57 (13), 5093 (2016).</li><li>Cortes, 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=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926-2932 (2015).</li><li>Bernal, A., Parel, J. -. M., Manns, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Posterior+Zonular+Fiber+Attachment+on+the+Anterior+Hyaloid+Membrane.">Evidence for Posterior Zonular Fiber Attachment on the Anterior Hyaloid Membrane.</a> Invest Ophthalmol Vis Sci. 47 (11), 4708 (2006).</li><li>Kammel, R., Ackermann, R., Mai, T., Damm, C., Nolte, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+Lenses+in+a+Lens+Stretcher.">Pig Lenses in a Lens Stretcher.</a> Optom Vis Sci. 89 (6), 908-915 (2012).</li><li>Hahn, 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=Measurement+of+Ex+Vivo+Porcine+Lens+Shape+During+Simulated+Accommodation,+Before+and+After+fs-Laser+Treatment.">Measurement of Ex Vivo Porcine Lens Shape During Simulated Accommodation, Before and After fs-Laser Treatment.</a> Invest Ophthalmol Vis Sci. 56 (9), 5332-5343 (2015).</li><li>D'Antin, J. C., Cortes, L. P., Montenegro, G. A., Barraquer, R. I., Michael, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+portable+manual+stretching+device+to+simulate+accommodation.">Evaluation of a portable manual stretching device to simulate accommodation.</a> Acta Ophthalmol. 93 (255), (2015).</li><li>Pierscionek, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+response+of+human+lenses+to+stretching+forces.">Age-related response of human lenses to stretching forces.</a> Exp Eye Res. 60 (3), 325-332 (1995).</li><li>Marussich, 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=Measurement+of+Crystalline+Lens+Volume+During+Accommodation+in+a+Lens+Stretcher.">Measurement of Crystalline Lens Volume During Accommodation in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 56 (8), 4239 (2015).</li><li>Martinez-Enriquez, E., P&#233;rez-Merino, P., Velasco-Ocana, M., Marcos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OCT-based+full+crystalline+lens+shape+change+during+accommodation+in+vivo.">OCT-based full crystalline lens shape change during accommodation in vivo.</a> Biomed Opt Exp. 8 (2), 918-933 (2017).</li></ol>

AB tem participação no Bioniko Consulting LLC.

Nós desenvolvemos um novo método para fornecer uma maneira precisa e eficiente de estudar a capacidade de alojamento da lente, utilizando um mecanismo de fixação de duplo-peça para acoplar a maca para a amostra. Durante o alojamento, a lente relaxa, e o diâmetro diminui em resposta ao relaxamento de fibras zonular1,2,4,19. O método centra-se sobre este fenómeno de aperto e controlando a...

A acomodação é o processo pelo qual o olho humano é capaz de ajustar dinamicamente a forma de seu cristalino para ver objetos a distâncias longe ou perto, em foco. Acomodação é um processo intrinsecamente biomecânico. Após estímulo neural, os músculos ciliares produzem uma força sobre o corpo ciliar e das fibras de zonule que atribuem a circunferência da lente da cápsula1,2. Embora existam diferentes teorias atrás a biomecânica de alojamento, a mais amplamente aceita é a hipótese de Helmholtz. De acordo com a hipótese, a lente está em um estado natural e esticado, correspondente à forma mais fina da lente que é ideal para o foco de objetos distantes. Para alterar o foco para objetos mais próximos, contraem os músculos ciliares e as fibras zonular estão relaxadas. Por sua vez, engrossa a lente, aumentando a curvatura da superfície anterior e posterior. Isso corresponde a um aumento no poder dióptrico que é necessário para a visão, portanto, uma menor distância focal1de perto.
A capacidade de acomodar é comprometida ao longo do tempo através de uma condição chamada presbiopia. Afetando todo mundo por 50 anos, presbiopia faz com que o olho não é possível alterar dinamicamente o foco de longe para fechar distâncias3. Para combater a presbiopia, métodos atuais são passivos incluindo lentes bifocais e lentes corretivas. Ao mesmo tempo aumentar a habilidade de focalizar objetos próximos em alguns aviões, tais tratamentos passivos não é possível restaurar a capacidade dinâmica de foco da lente,4,5. A fim de tratar a presbiopia eficientemente, ou possivelmente impedi-lo, há uma necessidade contínua para entender melhor o alojamento.
Para estudar a lente alojamento, um número de dispositivos foram desenvolvido para simular o fenômeno ex vivo4,6,7,8,9. Foram introduzidos, para monitorar o alongamento da lente através de de forças centrífugas8discos girando. Para replicar mais fielmente o fenômeno, lente alongamento dispositivos foram gradualmente introduzidos e inovou. Usando uma lente maca, Manns et al. caracterizada a força necessária para acomodar a lente enquanto correlacionando tal a potência da lente e o diâmetro equatorial9. Entendimento atual é que a lente endurece com a idade, resultando em uma mudança reduzida em forma de lente, em resposta a uma força igual a partir do corpo ciliar3,10,11,12.
Macas de lente atual muitas vezes envolvem uma configuração complexa, implementando eletrônica e taxas de alongamentos programáveis e requer o cadáver inteiro globo ocular6,7,10,13. Esta exigência aumenta o custo por experiência de mais de US $500,00 por olho e disponibilidade de amostra. Aqui nós apresentamos um método para replicar a lente alojamento a baixo custo, como o olho posterior totaliza cerca US $200,00. Pouco menos sofisticado do que muitos dispositivos usados hoje, a técnica é muito mais custo-eficaz e adotáveis sem comprometer os resultados. Este método é centrado em torno de uma maca de lente manual (MLS), representada na Figura 1e usa um sistema exclusivo de fixação sobre as fibras zonular e um método de torção radial para expandir o diâmetro da lente. A precisão fisiológica do protocolo é validada pelas conclusões do Bernal et al., que estudou o caminho pelo qual as fibras zonular anteriores e posteriores estão ligadas para a cápsula de lente14. Usando o design de sapatos personalizados, que exigem apenas a lente, zonule e corpo ciliar, objetivamos estudar biomecânica lente replicando Alojamento fisiológico.

<table border="0" cellpadding="0" cellspacing="0" style="width:856px;" width="856">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Manual Lens Stretcher</td>
			<td style="width:233px;">Bioniko</td>
			<td style="width:132px;">MLS</td>
			<td style="width:300px;">Different animal species will require different shoe sizes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Porcine Eye Samples</td>
			<td>George G. Ruppersberger; slaughterhouse</td>
			<td>N/A</td>
			<td>Whole eyeballs were obtained</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Eye Samples</td>
			<td>The National Disease Research Interchange</td>
			<td>N/A</td>
			<td>Posterior poles without corneas were ordered</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors (5 1/2&#39;&#39; Straight)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Forceps (4 1/2&#39;&#39;)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iPhone 6s</td>
			<td>Apple</td>
			<td>N/A</td>
			<td>Any imaging system with ~0.1 mm resolution will work</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hypochorite</td>
			<td>Clorox</td>
			<td>Clorox Regular-Bleach</td>
			<td>Any disinfectant will work</td>
		</tr>
	</tbody>
</table>

simulating the mechanics of lens accommodation via a manual lens stretcher

O objetivo do presente protocolo é imitar a biomecânica de acomodação fisiológica de uma maneira custo-eficiente e prática. Alojamento é alcançado através da contração do corpo ciliar e relaxamento das fibras zonule, que resulta em espessamento da lente necessário para visão de perto. Aqui, apresentamos um romance, um método simples, no qual alojamento é replicado por enrijecer as zónulas ligadas da cápsula da lente através de um esticador de lente manual (MLS). Esse método monitora o alongamento radial alcançado por uma lente quando submetido a uma força consistente e permite uma comparação de acomodar lentes, que podem ser esticadas, a não-acomodando lentes, que não podem ser esticadas. Importante, os casais de maca para as zónulas diretamente e não para a esclera do olho, exigindo apenas a lente, zónulas e corpo ciliar ao invés da amostra de todo o globo. Esta diferença pode diminuir significativamente o custo de aquisição de lentes de dador cadáver cerca de 62% em relação a aquisição de todo o globo.

Olhos de suínos, uma amostra comum para estudar a presbiopia através de lente alongamento4,15, obtiveram-se, (n = 10) de um matadouro local e este protocolo foi utilizado para observar a capacidade de alojamento das lentes. Figura 5 A mostra a comparação da lente suínos antes e após alongamento através da MLS. Houve um aumento de 0,07 mm média 0,19 ± raio de lente quando esti...

Watch this Scientific Journal Video about Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher at JoVE.com

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

Simulando a mecânica de lente Alojamento através de uma lente Manual esticador

Apresentamos um método eficiente de estudar Alojamento lente usando uma maca de lente manual. A imita protocolo fisiológico Alojamento puxando as zónulas ligado ao redor da cápsula da lente, desse modo, estendendo-se a lente.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

simulating-mechanics-lens-accommodation-via-manual-lens

Research

JoVE Journal

Bioengineering

6.7K visualizações.  University of Maryland. Apresentamos um método eficiente de estudar Alojamento lente usando uma maca de lente manual. A imita protocolo fisiológico Alojamento puxando as zónulas ligado ao redor da cápsula da lente, desse modo, estendendo-se a lente.

Vídeo: Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). We demonstrate in this video article how blast TBI-relevant impulsive pressurization is applied to the neuronal cells in vitro. This is achieved by using well-controlled pressure pulse created by a specialized Kolsky bar device, with complete pressure history within the cell pressurization chamber recorded. Pressurized neuronal cells are inspected immediately after pressurization, or further incubated to examine the long-term effects of impulsive pressurization on neurite/axonal outgrowth, neuronal gene expression, apoptosis, etc. We observed that impulsive pressurization at about 2 MPa induces distinct neurite loss relative to unpressurized cells. Our technique provides a novel method to investigate the molecular/cellular mechanisms of blast TBI, via impulsive pressurization of brain cells at well-controlled pressure magnitude and duration.

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate the molecular/cellular mechanisms of blast-induced traumatic brain injury.

Pressurização impulsivo de células neuronais de Estudos Traumático Brain Injury

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). ...

Bioengenharia

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Metodologia Elastografia por Ressonância Magnética para a Avaliação de engenharia de tecidos Crescimento Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

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 ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

A lignina Down-regulação da<em&gt; Zea mays</em&gt; Via dsRNAi e Klason Análise lignina

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Determinação do Potencial Zeta de nanopartículas de translocação através de velocidades por meio de um Tunable Nanopore: Utilizando partículas modificadas-ADN como um Exemplo

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Padronização da geometria de colônias de células-tronco embrionárias humanas em substratos compatíveis para controlar a mecânica de nível de tecido

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Transplante de um Patch Bioimpresso 3D em um modelo murino de infarto do miocárdio

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Sondando a mecânica do conjunto de miosina em feixes de filamentos de actina usando pinças ópticas

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

Triphosforilação química de Oligonucleotídeos

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple and practical solution. Therefore, a more feasible cerebral organoid protocol is proposed in the present study. To solve the unavoidable inconvenience in medium change and organoid transfer in the early stage, the current research optimizes the operation technology by applying the engineering principle. A soft light lamp was adopted to laterally illuminate the embryoid body (EB) samples, allowing the EBs to be seen by the naked eye through the enhanced diffuse reflection effect. Using the principle of secondary flow generated by rotation, the organoids gather toward the center of the well, which facilitates the operation of medium change or organoid transfer. Compared to the dispersed cell, the embryoid body settles faster in the pipette. Using this phenomenon, most of the free cells and dead cell fragments can be effectively removed in a simple way, preventing EBs from incurring damage from centrifugation. This study facilitates the operation of cerebral organoid culture and helps to promote the application of brain organoids.

Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination

Cerebral organoids provide unprecedented opportunities for studying organ development and human disease pathology. Although great success has been achieved with cerebral organoid culture systems, there are still operational difficulties in applying this technology. The present protocol describes a cerebral organoid procedure that facilitates medium change and organoid transfer.

Facilitando a cultura organoide cerebral através da iluminação lateral da luz suave

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple ...