Os autores gostariam de agradecer do fundo e departamento de cirurgia bolsa de pesquisa da Southern Illinois University, School of Medicine start-up; ea concessão Memorial Medical Foundation.

1. Recuperação Cirúrgica Nota: Uma aprovação IRB foi obtida para coletar o coto do LCA durante a cirurgia de reconstrução do LCA. Isenção IRB foi dado como o coto ACL usado geralmente eram descartados como lixo cirúrgico. 20 pacientes foram usados ​​para coletar o coto do LCA. <ol><li> Administrar anestesia geral e antibióticos pre-operatórios, conforme o protocolo da instituição para o paciente. </li><li> Aplicar torniquete e inflar 100 mg acima da pressão arterial sistólica. </li><li> Situar o paciente em decúbito dorsal. Faça uma incisão horizontal ântero-lateral com o joelho em flexão de 30-45 ° para um portal de 5 mm e inserir a trocar artroscópica e câmera na bolsa suprapatelar em 30 °. </li><li> Realize uma artroscopia diagnóstica de rotina. </li><li> Digite um entalhe e debridar sinóvia membranoso sobre o ACL rasgado. </li><li> Use uma máquina de barbear 4,5 mm e ablator para identificar o coto do LCA. </li><li> Use um bico de pato reta amargo para coletar SPE múltiplacimens do coto ACL restantes. </li><li> Manter a amostra em solução salina normal, num recipiente estéril. </li><li> Enviar amostras de patologia para decodificação e entrega final para a instalação do laboratório. </li><li> Recuperar paciente e executar cuidados pós-operatórios, conforme o protocolo da instituição. </li></ol> 2. Digestão do tecido e HACL Isolamento <ol><li> Transferir o tecido de leishmaniose humana recebido de patologia para uma placa de Petri com solução salina estéril / PBS (Tampão fosfato salino). </li><li> Picar o tecido, utilizando uma tesoura esterilizada em 1-2 mm 3 pedaços e lavar o tecido moído com uma solução salina, pelo menos, 3 vezes. </li><li> Digerir o tecido moído com 0,4% de colagenase em DMEM/F-12 (50/50, 1x), suplementado com 10% de soro fetal bovino (FBS) e 1% de Penicilina / Estreptomicina para 4-6 horas a 37 ° C. </li><li> Centrifugar as células a 1000 g durante 5 min e, em seguida, voltar a suspender as células no meio. </li><li> Lavar as células com o meio de 2-3 vezes e, em seguida, seed / cultura na T-25 frascos para 2-3 dias. </li></ol> 3. Expansão celular, congelamento e descongelamento <ol><li> Use de um microscópio de luz para visualizar as células em cultura durante 2-3 dias num frasco T-25. </li><li> Manter as células a 37 ° C no DMEM/F-12 (50/50, 1x), suplementado com 10% de soro fetal bovino (FBS) e 1% de Penicilina / Estreptomicina (P / S). Nota: Altere a mídia quando as células mostram adesão e morfologia normal. Use de um microscópio de luz para observar as células. </li><li> Lave as células confluentes com PBS no dia 7 (~ 1,3 X 10 6 células / frasco). Adicionar tripsina (0,5 g / L) e incuba-se as células a 37 ° C durante 5 min. Adicionar mídia para células em suspensão para neutralizar a tripsina. Divida a mistura de células de mídia entre três T-25 frascos. </li><li> Lave as células confluentes com PBS, adicionar tripsina (0,5 g / L), e ressuspender-los em meio contendo DMSO a congelação, de FBS e o meio completo (DMEM/F-12 Médio + FBS a 10% + 1% P / S) no proporção 01:02:07. </li><li> Lojaos frascos criogénicos a -80 ° C, se as células serão usados ​​dentro de um mês, e em azoto líquido, se as células será armazenada por períodos mais longos. </li><li> Descongelar os frascos num banho de água a 37 C ° para reutilização. Nota: Normalmente as células 90-95% sobreviver. </li></ol> 4. Fabricação de engenharia de tecidos 2-D Poly láctico-co-glicólico Ácido (PLAGA) Scaffold <ol><li> Dissolve-se 1 g de PLAGA em 12 ml de diclorometano num frasco de cintilação de 20 ml e vortex a solução durante 8 horas a uma velocidade constante (800 rpm). Nota: A descrição detalhada em 15 de Gupta et al. </li><li> Transferir a solução dissolvida a uma placa de Petri de vidro forrado com papel de proteção fluorados. </li><li> Colocar a placa de Petri, sob uma câmara de vácuo durante 30 min. Deixar a placa de Petri, a -20 ° C durante a noite e, em seguida, à temperatura ambiente. Colocar as placas de Petri em liofilizador para assegurar a completa evaporação do solvente para obtenção de películas finas de polímero. </li><li> Coloque o fi polímerolms num exsicador durante 24 horas. </li><li> Cortar os filmes de polímeros em 12 mm de diâmetro discos circulares e armazená-los num exsicador até ser necessário. </li></ol> 5. Microscopia Eletrônica de Varredura (MEV) <ol><li> Lavar as células (50.000 células / disco) semeado na TCPS controle eo andaime PLAGA com PBS sete dias posteriores à semeadura. </li><li> Use 1,5% de glutaraldeído em tampão cacodilato 0,1 M para corrigir as células e 2,5% OsO 4 em tampão cacodilato 0,1 M para a pós-fixação. </li><li> Lave as células fixadas com tampão de cacodilato 0,1 M. Secam-se as células fixadas utilizando desidratação série etanol (50%, 70%, 80%, 90% e 100%) durante 15 min cada, e ainda mais seco em hexametildisilazano (HMDS) durante a noite. </li><li> Ventilação da câmara de SEM sistema de revestimento por pulverização com válvula de botão e levantar a placa superior. </li><li> Coloque as amostras secas na câmara. Abaixe a placa superior. </li><li> Desligue o botão de controle para a bomba para começar a evacuar a câmara. </li><li> Válvula de escape aberta árgon. Esperepara o vácuo cair para 0,05 mbar. </li><li> Brasão das amostras secas com uma camada fina (0,13 nm) de ouro / paládio. </li><li> Retorne a válvula de escape de argônio para sua posição fechada. </li><li> Desligue o botão de controle para fora. </li><li> Alivie a câmara com válvula de botão e levantar a placa superior. </li><li> Remover as amostras e armazená-los em um dessecador por 24 horas. </li><li> Observe as amostras sob um microscópio eletrônico de varredura. </li></ol> 6. Imunofluorescência Coloração <ol><li> Lavar as células (50.000 células / disco) semeado na TCPS controle eo andaime PLAGA com PBS sete dias posteriores à semeadura. </li><li> Fixar as células com 70% de etanol (frio) por 10 min. </li><li> Incubar as células fixas à temperatura ambiente com 1% de Albumina de Soro Bovino (BSA) em PBS com 0,05% de Triton X-100 durante 20 min. </li><li> Mergulhar as amostras em 1% de Tween à temperatura ambiente durante 20 min. </li><li> Adicionar monoclonal de ratinho anti-actina-β anticorpo (1:400) e incuba-se as células OvernighT, a 4 ° C. </li><li> Lavar as células com 0,05% de Tween. Adicionar anticorpo secundário (anticorpo de cabra anti-rato F (ab &#39;) 2 fragmentos de IgG conjugado com uma sonda fluorescente, 1:400) para as células e incubar durante 1 hora à temperatura ambiente. </li><li> Lavar as células com PBS. Mancha as células com mancha nuclear e montar usando 80% de glicerol. </li><li> Observe as células em um microscópio confocal. </li></ol>

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

O objetivo principal deste estudo andaime hACL/2D era usar as células obtidas em um patch para aumentar o reparo primário de lágrimas ACL parciais. Tratamento não-cirúrgico de lesões do LCA parciais podem incluir um curto período de imobilização, órtese, um programa de reabilitação progressiva, e as avaliações de acompanhamento regulares 13,16,17. No entanto, muitos estudos mostraram que o tratamento conservador em atletas tem sido associado com resultados pobres e falha. Buckley et al....

O ligamento cruzado anterior (LCA) é um ligamento intra-articular comumente lesionado do joelho. Aproximadamente 200.000 (LCA) são relatados anualmente nos Estados Unidos. Mais de 75% dos pacientes que sofreram lesão do LCA opt para a cirurgia reconstrutiva ortopédico 1,2,3,4. A intervenção cirúrgica é muitas vezes indicada devido a um potencial de cura inerentemente pobre 3. Reconstrução ACL é tipicamente conseguida por meio de um auto-enxerto ou enxerto de tendão. Autotransplante e enxerto representam o padrão ouro para a reconstrução, como eles possuem altas taxas de sucesso, e reparo sutura primária, a outra opção de tratamento, mostrou taxas de falha de até 94% 5,6,7. Ruptura parcial do ACL representam 10% a 28% de todas as lesões do LCA 8. Em um estudo prospectivo, Noyes et al. Estima-se que 50% dos pacientes com lesões parciais que afetam mais da metade da ACL progrediu para completar ACL insuficiência depois nãotratamento cirúrgico 9. Outros estudos relatam instabilidade persistente e diminuição da função com menos de 30% dos pacientes capazes de retornar à sua pré-lesão nível de atividade 9,10,11,12,13. As opções de tratamento são limitadas e incluem modalidades conservadoras, encolhimento térmico do restante ACL, ou a reconstrução do LCA. Recentemente, tem havido um interesse crescente em reparo primário aumentada. Estas técnicas utilizam produtos biológicos para melhorar reparos sutura primária 14. Uma pesquisa recente tentou colher mesenquimais semelhantes HACL derivados de células-tronco para contornar as limitações do enxerto, 31,32 mas a validade e eficácia destas células ainda é desconhecido. A fonte ideal de células para aplicações de engenharia de tecidos parece ser não-mesechymal HACL derivadas de células de fibroblastos. A pesquisa atual está focada na identificação de um material de matriz adequada e fonte de células para o cadafalso engenharia. É procedimento padrão para um ACL rasgado para ser descartado como suresíduos rgical durante a cirurgia de reconstrução, no entanto, este ligamento danificado pode ser uma fonte de qualidade para a aquisição de células necessárias para desenvolver e melhorar um tecido ideal projetado substituição ACL. Nosso laboratório desenvolveu um protocolo para a expansão in vitro de células derivadas destas HACL colhidas. Usando uma matriz 2D engenharia infundido com células derivadas HACL, criamos um patch que pode potencialmente aumentar o reparo ACL parcial e fortalecer ligamentos rompidos.

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			<td height="21" style="height:21px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:507px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri-dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">08-757-103C</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP3994</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Collagenase</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">17018-029</td>
			<td>Store at 40C</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">DMEM/F-12</td>
			<td style="width:139px;">Cellgro</td>
			<td style="width:143px;">10-092-CV</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">10082</td>
			<td>Store at -800C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-602E</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Tubes- 15 ml</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430790</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T-25 flasks</td>
			<td style="width:139px;">BD Falcon</td>
			<td style="width:143px;">3013</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Trypsin-Versene mixture</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-161E</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP231-100</td>
			<td>Combustible liquid. Can cause skin, eye and respiratory tract irritation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryogenic vials</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430489</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">PLAGA</td>
			<td style="width:139px;">Purac Biomaterials</td>
			<td style="width:143px;">Purasorb PLG8523</td>
			<td>Store at -800C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCPS disks</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">12-545-82</td>
			<td></td>
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			<td height="23" style="height:23px;width:216px;">Dichloromethane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC36423-0010</td>
			<td>Possible cancer hazard. Store in a dry, cool place.</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Bytac paper</td>
			<td style="width:139px;">Saint gobin performance plastics</td>
			<td style="width:143px;">1420652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintillation vial</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>03-339-21G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A7906</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">mouse Anti-&#946;-actin antibody (10 Ab)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A5441</td>
			<td>Store at -200C</td>
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		<tr height="46">
			<td height="46" style="height:46px;width:216px;">goat-anti-mouse antibody (20 Ab)</td>
			<td style="width:139px;">Cell Signaling</td>
			<td style="width:143px;">4408</td>
			<td>Store at -200C</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Hoechst dye</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">14530</td>
			<td>Store at -200C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP229-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP2547-1</td>
			<td>Toxic by inhalation and if swallowed. Causes burns by all exposure routes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexamethyldisilazane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC43085-1000</td>
			<td>Flammable liquid and vapor. Causes burns by all exposure routes.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile Scissors</td>
			<td style="width:139px;">McKesson</td>
			<td style="width:143px;">25-716</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:139px;">Eppendorf</td>
			<td style="width:143px;">5804R</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light Microscope</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:143px;">CK40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water Bath</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:143px;">2845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:139px;">Labnet&#160;</td>
			<td style="width:143px;">VX-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Petri plates</td>
			<td style="width:139px;">fisher Scientific</td>
			<td style="width:143px;">S31473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acu-Punch</td>
			<td style="width:139px;">Acuderm Inc.</td>
			<td style="width:143px;">P1225</td>
			<td>Acu-Punch was used to cut 12mm disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cacodylate buffer</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">97068</td>
			<td>Flammable liquid, carcinogen and irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium tetraoxide</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">201030</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">T8787</td>
			<td>Harmful if swallowed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:139px;">Decon Labs</td>
			<td style="width:143px;">2705</td>
			<td>Keep away from heat, sparks, flame and other form of ignition.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">General/regional Anesthesia</td>
			<td style="width:139px;">Amphastar pharmaceuticals</td>
			<td style="width:143px;"></td>
			<td>1% Lidocaine, 0.25% Bupivacaine,Anesthetic agents for induction and maintenance</td>
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			<td height="21" style="height:21px;width:216px;">Antibiotics</td>
			<td style="width:139px;">Hospira</td>
			<td>0409-0805-01</td>
			<td>Ancef 1g i.v.</td>
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			<td height="21" style="height:21px;width:216px;">Arthroscopy trocar</td>
			<td style="width:139px;">Smith and Nephew</td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arthroscopy Camera</td>
			<td style="width:139px;">Smith and Nephew</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arthroscopic grasper and bitter</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4.5mm shaver</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Interference Screws</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td>stainless steel screws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sputter Coater</td>
			<td style="width:139px;">Polaron</td>
			<td style="width:143px;">E5400</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications

Prejuízo para o ACL é um problema comumente encontrado em indivíduos ativos. Mesmo lágrimas parciais deste intra-articular do ligamento do joelho levam a deficiências biomecânicas que prejudicam a função e estabilidade. As opções atuais para o tratamento das lesões do LCA parciais vão desde, o tratamento conservador não cirúrgico para várias opções cirúrgicas, tais como: modificação térmica, reparação de um único pacote, a reconstrução completa, ea reconstrução da parte danificada do ligamento nativo. Poucos estudos, se houver, têm demonstrado um método único para a gestão a ser consistentemente superior, e, em muitos casos, os pacientes continuam a demonstrar instabilidade persistente e outras co-morbidades. O objetivo deste estudo é identificar a fonte de células potencial de utilização no desenvolvimento de um patch de engenharia de tecidos que podem ser implementadas na reparação de uma ACL parcialmente rasgada. Um novo protocolo foi desenvolvido para a expansão de células derivadas de patients submetidos a reconstrução do LCA. Para isolar as células, tecidos HACL picada obtida durante a reconstrução ACL foi digerido numa solução de colagenase. A expansão foi realizada utilizando um meio de DMEM/F12 suplementado com 10% de soro fetal bovino (FBS) e 1% de penicilina / estreptomicina (P / S). As células foram então armazenadas a -80 ° C ou em azoto líquido num meio de congelamento que consiste em DMSO, de FBS e o meio de expansão. Após descongelação, as células derivadas HACL foram então semeados em um andaime de engenharia de tecidos, PLAGA (ácido láctico-co-glicólico poli) e controlo de cultura de tecidos de poliestireno (TCPS). Após 7 dias, SEM foi realizada para comparar a adesão celular à PLAGA versus o controlo TCPS. Morfologia celular foi avaliada utilizando técnica de imunofluorescência. SEM (Scanning Electron Microscope) micrografias demonstraram que as células cresceram e respeitados em ambas as superfícies plaga e PFC e foram confluentes sobre as superfícies inteiras por dia 7. Imunofluorescência mostrou morfológico normal, não-estressadospadrões AL em ambas as superfícies. Esta técnica é promissora para aplicações em LCA regeneração e reconstrução.

O modelo de trabalho para recuperação cirúrgica, a digestão do tecido e isolamento do ser humano Ligamento Cruzado Anterior (HACL) células derivadas é mostrado na Figura 1. As células migraram de explantes e aderiu à T-25 frascos. Estas células foram cultivadas durante 3 dias e, em seguida, foram visualizados sob um microscópio de luz (Figura 2). Uma monocamada confluente foi obtido no dia 7. A presença de células viáveis, saudáveis ​​indicou a recuperação bem sucedi...

Watch this Scientific Journal Video about Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications at JoVE.com

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Para aplicações futuras como um remendo para reparar ruptura parcial do ligamento cruzado anterior (LCA), LCA humano células derivadas foram isolados a partir de tecidos obtidos durante procedimentos reconstrutivos, expandidas in vitro e cultivados em engenharia de tecidos andaimes. Adesão celular e morfologia foi então realizada para confirmar a biocompatibilidade na superfície andaime.

Recuperação cirúrgica, isolamento e<em&gt; In vitro</em&gt; Expansão de humanos anteriores Cruciate células do ligamento derivados para aplicações em engenharia de tecidos

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

surgical-retrieval-isolation-vitro-expansion-human-anterior-cruciate

Research

JoVE Journal

Bioengineering

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

11.4K visualizações.  Southern Illinois University School of Medicine.  Para aplicações futuras como um remendo para reparar ruptura parcial do ligamento cruzado anterior (LCA), LCA humano células derivadas foram isolados a partir de tecidos obtidos durante procedimentos reconstrutivos, expandidas in vitro e cultivados em engenharia de tecidos andaimes. Adesão celular e morfologia foi então realizada para confirmar a biocompatibilidade na superfície andaime. 

Vídeo: Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Bioengenharia

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 ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritação dos olhos Test (EIT) para identificação de perigos de olho irritantes químicos usando reconstruída Humano Córnea-like epitelial (RHCE) Modelo Tissue

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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 ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Derreter a eletrofiação escrita de andaimes Poly(ε-caprolactone) tridimensional com morfologias controláveis para aplicações de engenharia de tecidos

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolamento de células-tronco mesenquimais do periósteo Alveolar humano e efeitos da vitamina D na atividade osteogênica do periósteo-derivado de células

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Dois métodos para Decellularization de tecidos vegetais para aplicações de engenharia de tecidos

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

Produção de Plasma Autólogo Rico em Plaquetas para Impulsionar a Expansão In Vitro de Fibroblastos Humanos

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Plataforma de Cultura de Órgãos do Segmento Anterior para Rastreamento de Lesões no Globo Aberto e Desempenho Terapêutico

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Tecido conjuntivo de engenharia humana derivada do fibroblasto para aplicações de triagem

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...