Os autores agradecem o apoio de novos prêmios NIH Director Innovator (1DP2 OD006499-01 a AA e 1DP2 OD006460-01 a AJE), e Abdulaziz King City para a Ciência e Tecnologia (UC San Diego Centro de Excelência em Nanomedicina). Gostaríamos de agradecer a Sra. Jessica Moore por seus comentários críticos sobre o manuscrito.

1. Síntese de acriloílo marcado por fluorescência-PEG-RGDS <ol><li> Reagir o péptido RGDS com éster carboximetil acriloil-PEG-succinimidil (APEG-SCM, PEG MW: 3400 g / mol) e di-isopropiletilamina NN (DIPEA) em razões molares 1.2:1:2 em sulfóxido de dimetilo (DMSO) sob atmosfera de árgon à temperatura ambiente durante a noite. </li><li> Confirmar conjugação por laser assistida por matriz tempo de dessorção / ionização de voo (MALDI-TOF) espectrometria de massa. Adicionar 1 ml de APEG-RGDS solução de reacção a uma mancha de amostra no alvo MALDI e seco. Prepara-se uma solução saturada de Universal MALDI Matrix em tetra-hidrofurano (THF) e agitar em vortex durante 1 min. Adicionar ~ pi 1 desta solução à mancha mesma amostra. Repita o procedimento para APEG-SCM para comparação. Carregar e analisar. O peso molecular de APEG-RGDS deve ser superior a APEG-SCM (Figura 2). </li><li> Para conjugar o fluoróforo, adicionar uma quantidade equimolar de Alexa Fluor 350 carboxílico (éster succinimydyl), dissolvidosnum volume mínimo de DMSO, a solução de reacção APEG-RGDS de 1,1 e reagir sob atmosfera de árgon à temperatura ambiente durante a noite. </li><li> Purificar APEG-RGDS-350 por diálise (PM 3500 Da) contra DI O 2-H, a 4 ° C, durante 48 horas à razão volumétrica 1.000:1, trocando dialisado pelo menos duas vezes por dia. </li><li> Congelar a secar purificado APEG-350-RGDS em um Labconco Freezone Plus ou sistema equivalente congelamento seco e armazenar a -20 ° C. </li></ol> 2. Preparação de um Molde de 2D e fabricação de um gel 2D com PEG-350 RGDS alternada Camadas <ol><li> Preparar lâminas de vidro hidrofóbicas. Colocar as lâminas de vidro limpas em um prato de vidro num forno de vácuo. Aquecer a 80 ° C durante 30 minutos para secar completamente as superfícies. Lugar prato com lâminas em um exaustor e adicionar 250 ul Sigmacote a cada slide, balançando suavemente por 30 segundos para a superfície revestimento inteiro. Enxaguar bem as lâminas revestidas com 100% de metanol, seguido de lavagem em água destilada, duas vezes por imersão durante 5 minutos em em least de 10 ml. </li><li> Cortar espaçadores de silicone (0,8 mm de espessura), com 10 milímetros de biópsia perfuradores. </li><li> Autoclavar os espaçadores de silicone e slides Sigmacote tratados com vidro. </li><li> Formular soluções para cada respectiva camada, em tubos de microcentrífuga individuais, de mistura de PEG diacrilato (PEGda) precursor (concentração final de 15% w / v) com quantidades diferentes de iodixanol (solução-mãe a 60% em água), para se obterem diferentes concentrações finais (por exemplo, 40%, 30%, 20% e 10%), completando o volume restante com tampão fosfato salino (PBS) para obter soluções de densidades calibradas. De uma maneira semelhante, por camadas alternadas, mistura APEG-RGDS-350 (concentração final de 8 mM) com PBS e iodixanol, para se obter várias concentrações (por exemplo, 35%, 25% e 15%). </li><li> Adicionar fotoiniciador (2-hidroxi-4&#39;-(2-hidroxietoxi)-2-metilpropiofenona, estoque 333mg/ml em N-vinil-pirrolidona) a cada solução por várias camadas (10 ul de solução de reserva por ml de cada solução de camada). Fotoiniciador is adicionados no final para evitar a polimerização antes estratificação dos géis no molde, uma vez que é sensível à luz. </li><li> Etapas subseqüentes deste protocolo será realizado em uma cabine de segurança biológica para assegurar a esterilidade. </li><li> Filtra-esterilizar cada solução utilizando uma seringa estéril ml e um filtro de 0,2 um. Montar a configuração do molde por ensanduichando o espaçador entre duas lâminas de vidro tratadas com Sigmacore e seguras com grampos colocação, conforme ilustrado na Figura 1. </li><li> Converter os géis em camadas, adicionando a solução mais denso (por exemplo PEGda com iodixanol 40%) em primeiro lugar, seguido por uma solução de menor densidade (por exemplo, APEG-RGDS-350 com iodixanol 35%). Repita as camadas alternadas de atingir várias camadas de composição desejada e densidades, como mostrado na Figura 1. </li><li> Irradiar o molde com luz de 365 nm, durante 3 min usando um portátil UVR-9000 da lâmpada. Permitir que os géis polimerizados curar durante 5 min. Remova os grampos, em seguida, levantar delicadamente a parte superior de vidroslides e do molde, os géis dGMP estratificadas permanecerá nos slides. Usando uma espátula estéril, colocar cuidadosamente os géis em tubo de 50 ml de PBS estéril contendo meio de cultura ou para a lavagem. </li><li> Lavar os géis polimerizados em PBS a 1.000:1 razão volumétrica, a troca de tampão de pelo menos duas vezes por dia, para remover o modificador de densidade, fotoiniciador, e um polímero que não reagiu. Alternativamente, PBS pode ser trocado com meio de crescimento celular. Armazenar os géis dGMP em PBS ou meio de crescimento para a cultura de células de ensaio descrito no Passo 3. </li><li> Para visualizar camadas alternadas, organizar DGMP gel (esses géis não poderá ser usado para cultura de células) ao longo de uma régua na bandeja de amostra de uma unidade de documentação de gel VersaDoc. Expor o gel em 350 nm, o tempo de exposição pode variar, dependendo da concentração do fluoróforo. Alternando bandas escuras e azul no hidrogel DGMP demonstrar a formação de camadas discretas da composição química diferente (Figura 3). </li></ol><ol><li> Para geles incorporando péptido RGDS, utilização de células dependentes da aderência, tais como mioblastos C2C12. </li><li> Insira cuidadosamente géis dGMP (armazenadas em PBS) nas cavidades de placas de 48 poços de cultura de células, utilizando um raspador de células estéril numa câmara de biossegurança. </li><li> Pré-aquecer o meio de crescimento (meio de Dulbecco modificado por Eagle ou DMEM suplementado com 10% v / v de soro fetal de bovino e de v / v de 1% 100x solução de penicilina-estreptomicina) e PBS num banho de água regulado a 37 ° C. </li><li> Lavar a placa de 60% confluentes (10 mm) de células C2C12 três vezes com PBS. Aspirar PBS e as células de colheita por adição de 1 ml de 0,25% de tripsina-EDTA e incubado a 37 ° C durante 2 min. Ressuspender as células em meio de crescimento e células de contagem. Semente da DGMP gel contendo a cultura de células bem com mioblastos C2C12 (20.000 células / cm 2). Incubar as células a 37 ° C em 5% CO2 / 95% de humidade relativa. Gentilmente meio de troca, após 4 horas, cuidado para não voltarmover levemente células aderidas. </li><li> Após 24 h, a fixação de mioblastos C2C12 sobre as camadas que contêm RGDS de géis dGMP pode ser confirmado por epifluorescência e microscopia de contraste de fase (Zeiss Axiovert 200). </li></ol>

<ol>
	<li>Place, E. S., Evans, N. D., Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+in+biomaterials+for+tissue+engineering.">Complexity in biomaterials for tissue engineering.</a> Nat. Mater. 8, 457-470 (2009).</li><li>Liu, V. A., Jastromb, W. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+protein+and+cell+adhesivity+using+PEO-terminated+triblock+polymers.">Engineering protein and cell adhesivity using PEO-terminated triblock polymers.</a> J. Biomed. Mater. Res. 60, 126-134 (2002).</li><li>Sharma, B., 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=Designing+zonal+organization+into+tissue-engineered+cartilage.">Designing zonal organization into tissue-engineered cartilage.</a> Tissue Eng. 13, 405-414 (2007).</li><li>Hahn, M. S., 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=Photolithographic+patterning+of+polyethylene+glycol+hydrogels.">Photolithographic patterning of polyethylene glycol hydrogels.</a> Biomaterials. 27, 2519-2524 (2006).</li><li>Kizilel, S., Sawardecker, E., Teymour, F., Perez-Luna, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+formation+of+covalently+bonded+hydrogel+multilayers+through+surface+initiated+photopolymerization.">Sequential formation of covalently bonded hydrogel multilayers through surface initiated photopolymerization.</a> Biomaterials. 27, 1209-1215 (2006).</li><li>Harley, B. A., 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=Design+of+a+multiphase+osteochondral+scaffold.+II.+Fabrication+of+a+mineralized+collagen-glycosaminoglycan+scaffold.">Design of a multiphase osteochondral scaffold. II. Fabrication of a mineralized collagen-glycosaminoglycan scaffold.</a> J. Biomed. Mater. Res. A. 92, 1066-1077 (2010).</li><li>Cuchiara, M. P., Allen, A. C., Chen, T. M., Miller, J. S., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+microfluidic+PEGDA+hydrogels.">Multilayer microfluidic PEGDA hydrogels.</a> Biomaterials. 31, 5491-5497 (2010).</li><li>Roam, J. L., Xu, H., Nguyen, P. K., Elbert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+protein+concentration+gradients+mediated+by+density+differences+of+poly(ethylene+glycol)+microspheres.">The formation of protein concentration gradients mediated by density differences of poly(ethylene glycol) microspheres.</a> Biomaterials. 31, 8642-8650 (2010).</li><li>Gleghorn, J. P., Lee, C. S., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+properties+of+laminated+alginate+gels+for+tissue+engineering+of+layered+structures.">Adhesive properties of laminated alginate gels for tissue engineering of layered structures.</a> J. Biomed. Mater. Res. A. 85, 611-618 (2008).</li><li>Karpiak, J. V., Ner, Y., Almutairi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+gradient+multilayer+polymerization+for+creating+complex+tissue.">Density gradient multilayer polymerization for creating complex tissue.</a> Adv. Mater. 24, 1466-1470 (2012).</li><li>Pierschbacher, M. D., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+attachment+activity+of+fibronectin+can+be+duplicated+by+small+synthetic+fragments+of+the+molecule.">Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule.</a> Nature. 309, 30-33 (1984).</li><li>Kaushik, G., Fuhrmann, A., Cammarato, A., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Situ+Mechanical+Analysis+of+Myofibrillar+Perturbation+and+Aging+on+Soft,+Bilayered+Drosophila+Myocardium."> In Situ Mechanical Analysis of Myofibrillar Perturbation and Aging on Soft, Bilayered Drosophila Myocardium.</a> Biophysical Journal. 101, 2629-2637 (2011).</li></ol>

Os autores não têm conflitos de interesses para divulgar.

DGMP é uma estratégia simples para a preparação de geles de várias camadas que não contam com a instrumentação dispendiosa. Este protocolo pode ser adaptado para a criação de andaimes que utilizam outros materiais biocompatíveis, como o colagénio e ácido hialurónico. Pequenas moléculas bioactivas, por exemplo a promoção de adesão celular de péptidos RGDS, pode ser amarrado à matriz polimérica para impedir a mistura de sinais entre as camadas. As proteínas podem ser encapsuladas em camadas distintas, sem a necessidade de conjugação química à medida que, dependendo do tamanho da malha da matriz, são menos propensas a se difundir através hidrogéis 10. Aqui foi utilizado iodixanol (Nycoprep), um modificador de densidade inerte, que tenha sido previamente utilizado para aplicações de células viáveis. Outros modificadores da densidade tais como a sacarose e dextrose pode também ser usado. Ao variar o tempo de sedimentação (t s), pode-se ajustar as interfaces entre duas camadas para produzir transições suaves ou afiado como necessário (tempo de sedimentação dá transições mais suaves) &lt;sup&gt; 10. Por exemplo, as transições suaves entre as camadas de gel dGMP poderia ser usado para gerar um gradiente contínuo de um sinal biológico para estudar processos celulares, tais como a quimiotaxia. O efeito do modificador de densidade na rigidez do gel é apresentado na Figura 5 para um 15% aPEGda gel; uma caracterização mais completa de rigidez e porosidade como uma função da concentração de iodixanol PEGda e está actualmente a ser avaliada. Embora a concentração PEGda neste exemplo é relativamente alta, observou-se um módulo de elasticidade de 60% maior do elástico em géis com iodixanol 30% em relação aos géis sem. A alteração na rigidez de gel pode ser ajustada para a concentração por modulação de macrómero a densidade de reticulação ou. Temos também aplicada a técnica de criar DGMP 3D utilizando géis de poliacrilamida de multicamadas e de precursores de PEG 10. Variando a concentração ou do grau de reticulação do polímero permite a variação estrutural noandaimes, os quais podem ser usados ​​para investigar o comportamento de células, tais como o crescimento e migração polarizada em 3D. Em resumo, DGMP é uma técnica flexível que pode ser aplicado para o fabrico de andaimes de 2D e 3D a partir de uma variedade de materiais biocompatíveis para uma ampla gama de aplicações de pesquisa biomédica e básica.

<table border="1"><tbody><tr><td> Reagente ou Instrumento </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Polietileno glicol succinimydyl carboximetilcelulose (a-PEG-SCM) </td><td> Laysan </td><td> 120-64 </td></tr><tr><td> Polyethelyene glicol diacrilato (PEGda) </td><td> Dajac Labs </td><td> 9359 </td></tr><tr><td> Arginina-glicina-ácido aspártico-serina (RGDS) </td><td> American Peptide </td><td> 49-01-4 </td></tr><tr><td> N, N - diisopropiletilamina (DIPEA) </td><td> Sigma </td><td> D125806 </td></tr><tr><td> Dimetil-sulfóxido (DMSO) </td><td> Sigma </td><td> D2438 </td></tr><tr><td> N, N-dimetilformamida (DMF) </td><td> Pescador </td><td> D119-4 </td></tr><tr><td> Tetrahidrofurano (THF) </td><td> Pescador </td><td> T397 </td></tr><tr><td> Cassete de diálise (3500Da) </td><td> Thermo Scientific </td><td> 66330 </td></tr><tr><td> Alexa Fluor 350 carboxílico succinimydyl </td><td> Life Technologies </td><td> A-10168 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> Espaçadores de silicone </td><td> Grainger </td><td> 1MWA4 </td></tr><tr><td> Biópsia socos </td><td> Acuderm </td><td> P1025 (10 mm) P850 (8 mm) </td></tr><tr><td> Fosfato de Dulbecco tamponado salino (DPBS) </td><td> Hyclone </td><td> SH30028 </td></tr><tr><td> Iodixanol (NycoPrep) </td><td> Pescador </td><td> NC9388846 </td></tr><tr><td> Ácido 2-hidroxi-4&#39;-(2-hidroxietoxi)-2-metilpropiofenona </td><td> Sigma </td><td> 410896 </td></tr><tr><td> Meio Dulbecco modificado por Eagle (DMEM) </td><td> Life Technologies </td><td> 11054 </td></tr><tr><td> De soro fetal bovino </td><td> Life Technologies </td><td> 10082 </td></tr><tr><td> Penicilina-estreptomicina </td><td> Life Technologies </td><td> 15140 </td></tr><tr><td> Mioblastos C2C12 </td><td> ATCC </td><td> CRL-1772 </td></tr><tr><td> MALDI </td><td> Bruker </td><td> N / A </td></tr><tr><td> RUV-9000 </td><td> Bayco </td><td> RUV-9000 </td></tr><tr><td> VersaDoc </td><td> Bio-Rad </td><td> N / A </td></tr></tbody></table>

density gradient multilayered polymerization (dgmp): a novel technique for creating multi-compartment, customizable scaffolds for tissue engineering

Matrizes de tecido complexos de cultura, em que os tipos e concentrações de estímulos biológicos (por exemplo, factores de crescimento, inibidores, ou moléculas pequenas) ou a estrutura de matriz (por exemplo, composição, concentração, ou rigidez da matriz) variar ao longo do espaço, de permitir uma vasta gama de investigações sobre a forma como estas variáveis ​​afetam a diferenciação celular, migração e outros fenômenos. O desafio importante na criação de matrizes de camadas é a manutenção da integridade estrutural de interfaces de camada sem difusão dos componentes individuais a partir de cada uma das camadas 1. Metodologias atuais para alcançar este incluem photopatterning 2-3, litografia 4, functionalization5 seqüencial, liofilização 6, microfluídica 7, 8 ou centrifugação, muitos dos quais exigem instrumentação sofisticada e habilidades técnicas. Outros dependem de ligação sequencial de camadas individuais, que podem levar à delaminação das camadas 9 </sup&gt;. DGMP supera estes problemas por meio de um modificador de densidade inerte tal como o iodixanol para criar camadas de diferentes densidades de 10. Uma vez que o modificador de densidade pode ser misturado com o pré-polímero ou qualquer molécula bioactiva, DGMP permite que cada camada de andaime para ser personalizado. Simplesmente variando a concentração do modificador de densidade impede a mistura das camadas adjacentes, enquanto eles permanecem aquosa. Polimerização único passo subsequente dá origem a uma estruturalmente contínuo multicamada de andaime, em que cada camada tem química distinta e propriedades mecânicas. O modificador de densidade pode ser facilmente removido com a lavagem suficiente sem perturbação das camadas individuais ou os seus componentes. Esta técnica é, portanto, bem adequado para a criação de hidrogéis de várias dimensões, formas e materiais. Um protocolo para a fabricação de um polietileno glicol 2D-gel (PEG), em que as camadas alternadas incorporar RGDS-350, está descrito a seguir. Usamos PEG bomo é biocompatível e inerte. RGDS, a adesão de células de péptido 11, é utilizado para demonstrar a restrição espacial de um sinal biológico, e a conjugação de um fluoróforo (Alexa Fluor 350) permite-nos distinguir visualmente as diversas camadas. Este procedimento pode ser adaptado para outros materiais (por exemplo, colagénio, ácido hialurónico, etc) e pode ser estendido para o fabrico de geles de 3D ​​10 com algumas modificações.

MALDI-TOF análise confirma a conjugação do péptido RGDS de acriloil-PEG (Figura 2). Gel de imagem revela alternada RGDS-350 (azul) as camadas após a fotopolimerização (Figura 3A). Como mostrado na Figura 3A, 2D tamanho DGMP gel pode ser variada em função do diâmetro dos moldes de silicone (10 mm, esquerda, 8 mm, para a direita), e, portanto, são facilmente adaptável para utilização em ensaios múltiplos - neste caso, para se ajustar um 48 placas de cultura de células bem (Figura 3B). Epifluorescência e microscopia de contraste de fase de mioblastos C2C12 cultivados sobre um gel DGMP mostra ligação selectiva em RGDS-350-camadas contendo PEG (Figura 4), ​​demonstrando a compartimentalização do peptídeo de adesão celular (RGDS). <img src="/files/ftp_upload/50018/50018fig1.jpg" alt="Figura 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50018/50018fig1highres.jpg"/> Figura 1. Molecular Weight análise por MALDI-TOF comparando APEG-SCM de APEG-RGDS obtidos após a conjugação do péptido RGDS. <img src="/files/ftp_upload/50018/50018fig2.jpg" alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/50018/50018fig2highres.jpg"/> Figura 2. Representação esquemática da fabricação DGMP gel. Após os gradientes são em camadas, que pode ser deixado em repouso por períodos de tempo variáveis ​​(t s) para criar interfaces graduados, seguidos por fotopolimerização. Géis estratificadas dGMP pode ser facilmente extraído do molde para uso posterior. <a href="https://www.jove.com/files/ftp_upload/50018/50018fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img src="/files/ftp_upload/50018/50018fig3.jpg" alt="Figura 3" /> Figura 3 a.) 2D géis de multicamadas obtidas após fotopolimerização fotografada usando 350 nm e brancas canais de luz de VersaDoc unidade de documentação de gel. Escala cinza revela camadas alternadas contendo RGDS em branco. B) Inserção de gel DGMP em 48 pratos de cultura de células bem. <img src="/files/ftp_upload/50018/50018fig4.jpg" alt="Figura 4" /> Figura 4. Contraste de fase Incorporada e imagem epifluorescência de mioblastos C2C12 cultivados em DGMP géis (barra de escala 50 mm). <img src="/files/ftp_upload/50018/50018fig5.jpg" alt="Figura 5" /> Figura 5. Efeito de iodixanol na superfície do gel. Elasticidade medições de força atômica microscopia de amostras de substratos estáticos PEG reticuladas utilizando métodos previamente estabelecidos com uma força de disparo 2 nN 12. * P &lt;0,05 e ** p &lt;0,01.

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Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Aqui descrevemos uma estratégia única para a criação de biocompatíveis, matrizes em camadas com interfaces contínuas entre camadas distintas para engenharia de tecidos. Tal um andaime poderia proporcionar um ambiente ideal para modular o comportamento personalizável célula por vários estímulos químicos, biológicos ou mecânicos

Gradiente de densidade de várias camadas de polimerização (DGMP): uma nova técnica para a criação de multi-compartimento, Andaimes customizáveis ​​para Engenharia de Tecidos

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

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Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

12.4K visualizações.  University of California, San Diego. Aqui descrevemos uma estratégia única para a criação de biocompatíveis, matrizes em camadas com interfaces contínuas entre camadas distintas para engenharia de tecidos. Tal um andaime poderia proporcionar um ambiente ideal para modular o comportamento personalizável célula por vários estímulos químicos, biológicos ou mecânicos

Vídeo: Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomérico Andaimes em Engenharia de Tecidos Arterial

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Bioengenharia

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Processamento de pós-produção de Fibras electrospun para Engenharia de Tecidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Andaimes auto-relato para cultura de células 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Um biorreator Novel de Cultivo de Alta Densidade de diversas comunidades microbianas

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

Fabricação baseada em vara de customizáveis ​​macias robóticos pneumáticas Gripper Aparelhos para a manipulação de tecidos delicados

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Neonatal Cardiac Scaffolds: Matrizes novas para Estudos Regenerativa

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

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 ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting da cartilagem e Analogs de tecido de pele utilizando um romance passiva unidade técnica de mistura para Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Um protocolo para Decellularizing Cochleae Mouse para engenharia de tecidos do ouvido interno

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Cultivo de células de mamíferos em andaimes tridimensionais do Peptide

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Gradiente de densidade de várias camadas de polimerização (DGMP): uma nova técnica para a criação de multi-compartimento, Andaimes customizáveis ​​para Engenharia de Tecidos

Gradiente de densidade de várias camadas de polimerização (DGMP): uma nova técnica para a criação de multi-compartimento, Andaimes customizáveis para Engenharia de Tecidos