Os autores desejam agradecer a Valerie Schroeder pela sua assistência com aspectos técnicos deste trabalho. Trabalhos relacionados com o desenvolvimento deste modelo foi apoiado por Cook Biotech, Inc. (West Lafayette, nos EUA).

a utilização de animais neste protocolo foi aprovada pela Universidade de Notre Dame institucional Cuidado Animal e usar o Comitê, seguido de regulamentares todos os requisitos e diretrizes e foi realizada em uma instalação que é credenciada pelo Associação para a avaliação e acreditação do laboratório Cuidado Animal (AAALAC), International.
 1. seleção de animais <ol><li>ratos Sprague Dawley isentos de organismos patogénicos adquirir de um fornecedor respeitável ou colônia de reprodução. 
	Nota: Muitas vezes, o veterinário facilidade ou o gestor pode direcionar o investigador para fontes apropriadas. Ratos de aproximadamente 250 g de massa corporal são adequados para este procedimento.</li>
	<li>Um dia antes da cirurgia, examinar os animais para ser usado para garantir que eles são clinicamente saudáveis. A este respeito, pesar o animal e avaliar a condição do corpo; Observe qualquer anormalidade na respiração, a presença de quaisquer feridas abertas ou massas e o nível de atividade geral do animal. Observe qualquer anormalidade no registro cirúrgica e considerar tais observações como critérios de exclusão potencial.</li>
</ol> 2. Anestesia e preparação do Animal para a cirurgia asséptica <ol><li>para analgesia, administrar 1.1-1.2 mg/kg de liberação sustentada de buprenorfina, por via subcutânea para o rato 2 h antes de cirurgia. aplicar a pomada oftálmica de ambos os olhos e administrar aquecido de 2 mL de solução salina fisiológica estéril por via subcutânea.</li>
	<li>Anestesiar o rato com uma dose intraperitoneal de cloridrato de cetamina (90 mg/kg) e xilazina (10mg/kg). Aplique uma pequena quantidade de lubrificante ocular de cada olho para evitar a dessecação da córnea. Uma vez que o animal &#8217; respiração s é constante e não existe qualquer resposta a uma pitada de dedo do pé firme, o rato atingiu um nível de anestesia suficiente para a cirurgia. Um nível aceitável de anestesia pode ser confirmado pela presença de fundo, respiração regular e a cor normal do olho (contra palidez, que pode sugerir inadequada perfusão cardiovascular).</li>
	<li>Raspar o abdômen ventral com um cortador comercial do púbis ao esterno e das margens laterais sobre o ventrum. Remover o cabelo cortado, aplicando uma toalha de papel úmida para a área de.</li>
	<li>Esfregue a área depilada com um Iodofor anti-séptico seguida de uma limpeza com etanol a 70%. Aplicar cirúrgicos para que uma janela para a incisão pode ser isolada de potencial contaminação.</li>
	<li>Don um avental cirúrgico estéril, uma máscara facial e uma touca de cabelo. Cuidadosamente Lave e esfregue as mãos com sabão e don luvas cirúrgicas estéreis.</li>
</ol> 3. Criação e reparo da Hérnia Ventral Abdominal <ol><li>antes da cirurgia, esterilizar instrumentos cirúrgicos por autoclave.</li>
	<li>Com um bisturi, faça uma incisão aproximadamente 3-4 cm vertical ao longo da linha mediana ventral até ao nível da musculatura abdominal. Levante a parede abdominal delicadamente com uma pinça e criar uma pequena abertura através da linea alba. Para proteger as vísceras subjacentes, inserir uma pinça através do orifício e na cavidade abdominal para servir como um guia de proteção conforme a parede muscular é incisada para criar um aproximadamente 2 cm x 2 cm espessura completo defeito, corte aproximadamente lateral de 1 cm de cada lado da linha média em ambas as extremidades do linea a incisão alba.</li>
	<li>Colocar uma seção de corte 2 x 2 cm de material ponte mecânica, 4 camadas SIS neste caso (Figura 1), sobre o defeito. 
	Nota: Como alternativa, uma subjacência abordagem pode ser usada em que seção do material é colocada na cavidade abdominal para cobrir o defeito do aspecto interior.</li>
	<li>Sutura dos quatro cantos da ponte materiais para os músculos da parede abdominal usando seda ou nylon 4-0 em um padrão de sutura interrompido simples. 
	Nota: O uso de sutura não absorvível permite fácil identificação do material implantado após colheita contra material de sutura absorvível.</li>
	<li>Fixe cada borda do material com três suturas adicionais espaçadas igualmente. Examinar a borda inteira circunferencial para garantir que não existem lacunas significativas que pode permitir a passagem das vísceras através da parede abdominal; e, se existem tais lacunas, colocar suturas adicionais para garantir ainda mais a margem de.</li>
	<li>Fechar o tecido subcutâneo com sutura de 4-0 em um padrão simples interrompido. Fechar a pele com grampos cirúrgicos, apesar de sutura aplicada em um padrão simples interrompido também pode ser utilizada.</li>
	<li>Não abandone o animal até que ele recuperou a consciência suficiente para recuperar a prostração esternal; e não retornam um animal que foi submetido a cirurgia para a companhia de outros animais até que recuperou totalmente da anestesia. Durante este tempo, manter o animal em uma área morna para evitar hipotermia.</li>
	<li>Examine animais diariamente que foram submetidos a este procedimento para garantir a completa recuperação e cicatrização normal. Remover grampos de pele com um removedor de grampos de 7-10 dias após a cirurgia.</li>
</ol> 4. Colheita do Material implantado <ol><li>consistente com as necessidades do estudo, eutanásia, com overdose de dióxido de carbono, administrada em uma câmara encher taxa de 10 a 30%, os animais em um tempo pré-determinado, muitas vezes 21 - 28 dias após a cirurgia.</li>
	<li>Usando ferramentas de necropsia padrão, abrir a pele e a parede abdominal e expor a área do implante. 
	Nota: Se as suturas não absorvíveis foram utilizadas para fixar o implante, o material pode ser identificado pelas suturas originais (Figura 2).</li>
	<li>Observar quaisquer observações patológicas brutas, incluindo quaisquer reacções adversas (por exemplo, deiscência da ferida, abscesso, seroma, hematoma) e extensão de aderência e tenacidade para as vísceras subjacentes.</li>
	<li>Remover do local do implante por um corte de uma ampla secção de tecido que inclui o implante. Colocar a amostra em um fixador, tais como formalina 10% tamponada neutro, em preparação para o processamento para exame histológico e caracterização.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+strength+comparison+of+small+intestinal+submucosa+body+wall+repair.">Tensile strength comparison of small intestinal submucosa body wall repair.</a> J. Surg. Res. 135 (1), 9-17 (2006).</li><li>Garcia-Moreno, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+host+tissue+response+and+peritoneal+behavior+of+composite+meshes+used+for+ventral+hernia+repair.">Comparing the host tissue response and peritoneal behavior of composite meshes used for ventral hernia repair.</a> J. Surg. Res. 193 (1), 470-482 (2015).</li><li>Soiderer, E. E., Lantz, G. C., Kazacos, E. A., Hodde, J. P., Wiegand, R. 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Y., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biomechanical+behavior+and+host+response+to+porcine-derived+small+intestine+submucosa,+pericardium+and+dermal+matrix+acellular+grafts+in+a+rat+abdominal+defect+model.">The biomechanical behavior and host response to porcine-derived small intestine submucosa, pericardium and dermal matrix acellular grafts in a rat abdominal defect model.</a> Biomaterials. 32 (29), 7086-7095 (2011).</li><li>Ma, J., Sahoo, S., Baker, A. R., Derwin, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+muscle+regeneration+with+a+dermis/small+intestinal+submucosa+scaffold+in+a+rat+full-thickness+abdominal+wall+defect+model.">Investigating muscle regeneration with a dermis/small intestinal submucosa scaffold in a rat full-thickness abdominal wall defect model.</a> J.Biomed.Mater.Res.Part B. 103 (2), 355-364 (2015).</li><li>Greca, F. H., Souza-Filho, Z. A., Giovanni, A., Rubin, M. R., Kuenzer, R. F., Reese, F. B., Araujo, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+porosity+on+the+integration+histology+of+two+polypropylene+meshes+for+the+treatment+of+abdominal+wall+defects+in+dogs.">The influence of porosity on the integration histology of two polypropylene meshes for the treatment of abdominal wall defects in dogs.</a> Hernia. 12 (1), 45-49 (2008).</li><li>Hiles, M., Record-Ritchie, R. D., Altizer, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+biologic+grafts+effective+for+hernia+repair?+A+systematic+review+of+the+literature.">Are biologic grafts effective for hernia repair? A systematic review of the literature.</a> Surg. Innov. 16 (1), 26-37 (2009).</li><li>Helton, W. S., Fisichella, P. M., Berger, R., Horgan, S., Espat, N. 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Um dos autores (CJ) é empregado pelo Cook Biotech, Inc., o fabricante de grau médico SIS.

Materiais para o reparo da hérnia abdominal ventral são de grande interesse, particularmente aqueles que fornecem uma ponte mecânica inicial e então são capazes de incorporar no tecido do hospedeiro. A este respeito, uma variedade de materiais têm sido investigados, incluindo SIS, porcino matriz dérmica acelular e Pericárdio Porcino. 6 , 7 estes materiais representam as matrizes extracelulares daqueles tecidos e atuam como andaimes no qual as células pod...

Hérnia de parede abdominal é um problema clínico comumente encontrado que pode ocorrer como resultado de defeito congênito, lesão traumática ou falhado fechamento das feridas cirúrgicas, envolvendo a parede abdominal. Reparação geralmente envolve o uso de um implante para reforçar a parede abdominal, com uma taxa reduzida de reincidência, observada em pacientes tratados com um implante em comparação com aqueles em quem a parede abdominal é simplesmente fechada com sutura. 1
Reparo cirúrgico de hérnia muitas vezes requer ponte mecânica do defeito com um material implantado. A este respeito, materiais sintéticos e naturais têm sido utilizados para o reparo da hérnia. Muitos materiais sintéticos são tipicamente não-absorvíveis e incluem poliéster de tereftalato de polietileno, polipropileno e politetrafluoretileno expandido, enquanto outros implantes sintéticos combinam um dos materiais não absorvíveis com um absorvente polímero, tais como polygalactine. 1 em contraste, implantes de materiais naturais são geralmente derivados fontes cadavéricos humanas ou animais. Os materiais naturais utilizados para reparo de hérnia são geralmente ricos em colágeno e atuam como um andaime para crescimento interno e a integração do tecido do hospedeiro.
O desejo de desenvolver e otimizar novos materiais para reparação de hérnia abdominal ventral requer um modelo animal adequado para avaliação de materiais do candidato. Estudos têm sido realizados em uma variedade de espécies, incluindo ovelhas,2 porcos, coelhos de3 ,4 ratos,5,6,7,8 e cães. 9 a vantagem de usar ratos para tais estudos é que eles oferecem um modelo facilmente manipulado disponível em uma variedade de linhagens puras para permitir o controle da variabilidade genética. Além disso, a menor total área de superfície corporal do rato em comparação com as outras espécies listadas acima possibilitar a avaliação de materiais que podem ser experimentais na natureza e, portanto, não abundante.  Além disso, reduzidos os custos associados com ratos contra outras espécies permitem capacidade relativamente maior de materiais do candidato tela para uso em reparação de hérnia abdominal ventral.  Considerações para materiais normalmente incluem a facilidade de manuseio pelo cirurgião, força, capacidade de incorporar no tecido do hospedeiro, biocompatibilidade e resistência à infecção.
Como exemplo, submucosa intestinal pequeno suíno (SIS) é um biomaterial natural que tem sido usado para uma grande variedade de indicações de reparação de tecidos, incluindo reparação de hérnias inguinais, abdominais e diafragmática ventrais. 10 , 11 , 12 SIS rapidamente incorporada ao tecido do hospedeiro e foi encontrada para ser geralmente resistentes à infecção, e então o material tem particular utilidade em campos contaminados. 13 , 14 no modelo de rato, SIS demonstrou facilidade de manipulação, boa resistência à tração, conforme determinado pelo teste de falha de tração uniaxial e promoção de crescimento interno de tecido em termos de deposição de colágeno e neovascularização. 6 , 7
Ponte cirúrgica exata do defeito é uma característica essencial de modelagem produtiva quando avaliando materiais de reparação de candidato. O animal deve ser suficientemente anestesiada e asséptica técnica estritamente seguida. Além disso, um defeito da parede abdominal do tamanho padrão deve ser cuidadosamente criado e em seguida em ponte de forma suficientemente protege o material para as margens do defeito. 8 este protocolo fornece um método padrão a ser seguido para criação cirúrgica e reparação de uma hérnia abdominal ventral usando SIS como exemplo ponte material em um rato.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Isoflurane (Isoflo)</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">17579</td>
			<td style="width:167px;">gas anesthetic</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Optixcare Eye Lubricant</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td style="width:119px;">OPX4240</td>
			<td>Ocular lubricant</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Oster clippers</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">6092</td>
			<td>hair clippers</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Betadine surgical scrub</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">1618</td>
			<td>antiseptic iodophor</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Sterile scalpel</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">329</td>
			<td>#10 scalpel</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">9&#34; x 12&#34; disposable surgical drape</td>
			<td style="width:71px;">Braintree Scientific, Inc.</td>
			<td style="width:119px;">SP-RPS</td>
			<td>Surgical drape</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">4-0 nylon suture</td>
			<td style="width:71px;">Braintree Scientific, Inc.</td>
			<td style="width:119px;">SUT 812</td>
			<td>Suture material</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">4-0 Absorbable Suture</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">29242</td>
			<td>Synthetic absorbable suture</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">9 mm Autoclips and applier</td>
			<td style="width:71px;">Braintree Scientific, Inc.</td>
			<td style="width:119px;">ACS KIT</td>
			<td>Surgical staples for closure of skin incision</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Torbugesic/analgesic</td>
			<td style="width:71px;">Henry Schine Animal Health</td>
			<td align="right" style="width:119px;">12084</td>
			<td>Butorphanol tartarate for post-operative analgesia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Small intestinal submucosa (SIS)</td>
			<td style="width:71px;">Cook Biotech, Inc.</td>
			<td style="width:119px;"></td>
			<td>Implant material for bridging experimental abdominal wall defect</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rats</td>
			<td style="width:71px;">Harlan, Inc.</td>
			<td style="width:119px;">Sprague Dawley</td>
			<td>Animal for hernia modeling</td>
		</tr>
	</tbody>
</table>

use of a rat model to study ventral abdominal hernia repair

Hérnia abdominal ventral é uma condição clínica relativamente comum que às vezes requer herniorraphy (reparo cirúrgico). O reparo da hérnia abdominal ventral normalmente requer a implantação de um material para servir como uma ponte mecânica através do defeito na parede abdominal. Biomateriais, tais como a submucosa intestinal pequeno suíno (SIS), também servem como uma estrutura para o crescimento da célula para o implante e naturalmente podem incorporar o tecido do hospedeiro. Desenvolvimento de tais materiais de reparação beneficia da utilização de modelos animais em que os defeitos da parede abdominal experimental são facilmente criados em são favoráveis para reparar de forma reproduzível. O método oferecido aqui descreve criação cirúrgica e reparo da hérnia abdominal ventral em um modelo do rato. Quando o SIS é usada para reparar uma hérnia abdominal ventral experimental neste modelo, que é rapidamente incorporada tecido do hospedeiro dentro de 28 dias de implantação. Histologicamente, a incorporação do seu material implantado no tecido do hospedeiro caracteriza-se por uma resposta fibrovasculares robusta. Futuros aperfeiçoamentos e aplicações do modelo de hérnia abdominal rato provavelmente podem envolver animais diabéticos e/ou obesos como um meio para mais pròxima imitar comuns co-morbidades do homem.

Por 28 dias após o implante, SIS normalmente demonstra boa incorporação no tecido do hospedeiro (Figura 2). Na maioria dos casos, há SIS residual aparente, embora o tecido deve ser explorado, muitas vezes, para identificar o material implantado. Análise histológica confirmou as observações de patológicas brutas, ilustrando a incorporação de bom tecido com principalmente tecidos fibrovasculares e muito pouca mana residual observado (Figura 3). E...

Watch this Scientific Journal Video about Use of a Rat Model to Study Ventral Abdominal Hernia Repair at JoVE.com

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Este manuscrito descreve métodos associados com a criação e a reparação de um defeito da parede abdominal ventral (hérnia). Este modelo pode ser usado para estudar estratégias de reparação, tais como aqueles que usam materiais implantados. Neste manuscrito, reparo da hérnia experimental com suínos submucosa intestinal pequena é apresentado como um exemplo.

Utilização de um modelo do rato para estudar hérnia Abdominal Ventral

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

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9.1K visualizações.  University of Minnesota. Este manuscrito descreve métodos associados com a criação e a reparação de um defeito da parede abdominal ventral (hérnia). Este modelo pode ser usado para estudar estratégias de reparação, tais como aqueles que usam materiais implantados. Neste manuscrito, reparo da hérnia experimental com suínos submucosa intestinal pequena é apresentado como um exemplo.

Vídeo: Use of a Rat Model to Study Ventral Abdominal Hernia Repair

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

A Pele Tridimensional Humanos Reconstruir Modelo: uma Ferramenta para o Estudo da Pele Normal e Progressão Melanoma

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Bioengenharia

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 ...

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges1,2. These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production3. In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (&lambda;=532 nm, Fluence~110 J/cm2, spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 &plusmn; 6 kPa, (n=30). The adhesion strength drops to 2 &plusmn; 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.

Sutures are usually needed to repair tissue during surgical procedures. However, their application can be problematic as they are invasive and may damage tissue. The fabrication and application methods of a novel tissue adhesive are here reported. This adhesive film is laser-activated and does not require the use of sutures.

Fabricação e Aplicação de Rosa Bengala-quitosana Films em Reparo de Tecidos Laser

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

We describe the design of the &#x201C;quick-linker&#x201D; device for easier orthotopic rat liver transplantation.

Dispositivos de fabricação e instrumentos para facilitar a Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

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

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

Atímica Rat Modelo de Avaliação da Engineered Ligamento Cruzado Anterior Enxertos

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

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

In Vitro permeação de Ferritinas FITC-carregados através de um Rat barreira sangue-cérebro: um modelo para estudar a Entrega de Nanoformulated Moléculas

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

aplicações de<em&gt; In Vivo</em&gt; Teste Funcional do Rato tibial anterior para a Avaliação da engenharia de tecidos Repair Músculo Esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

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

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

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

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

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

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

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

O uso de um ensaio de β-lactamase-baseado titulometria Biosensor para detectar interações biomoleculares

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

Um protocolo eficiente e reprodutível para distração osteogênese em um modelo de rato, levando a um fêmur regenerada funcional

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...