Este trabalho foi financiado pela Fundação de Pesquisa Nacional de Cingapura administrado por um dos seus centros de investigação de excelência, o Instituto mechanobiology, Cingapura. MFAC é apoiada pela Agência da Ciência, Tecnologia e Pesquisa (Singapura) e Agência Nacional de Investigação (França) programa conjunto com o número 1122703037. BKKT projeto é apoiado pelo Instituto mechanobiology. Agradecemos ao Sr. Daniel HC Wong para o manuscrito e Ms. Amanhecer JH Neo para auxiliar na produção de vídeo de leitura de prova.

1. Preparação de Soluções de polielectrólito <ol><li> Purifica-se o quitosano, conforme detalhado na Liao et ai. Resumidamente, criar uma solução a 1% de quitosano em 2% (w / v) (v / v) de ácido acético e de vácuo filtro usando papel de filtro de grau 93. Neutraliza-se o filtrado utilizando NaOH 5M até que o pH estabilizou a 7. Centrifugar o quitosano precipitado a 1.200 xg durante 10 min. Decantar o sobrenadante e adicionar água desionizada para lavar o quitosano. Repetir o passo de lavagem e centrifugação mais duas vezes. Congelar a quitosana precipitado a -80 ° C e liofilizar O / N para se obter a forma purificada. Armazenar quitosana purificada num armário desumidificado. </li><li> Pesar 1 g de quitosana purificada em um estéril de cultura de tecidos prato. Coloque a quitosana no prato de cultura de tecidos tão perto quanto possível a luz UV na câmara de segurança biológica e expor à luz UV durante 15 min. Utilizando uma pinça estéril, colocar a quitosana esterilizado em um recipiente de vidro. Dissolver quitosana usando filtrada 0.15M de ácido acético até uma concentração final entre 0,5% e 1% (w / v). </li><li> Pesar 0,1 g de sal de sódio do ácido algínico e dissolver em 10 mL de água desionizada (DDI) de água destilada para se obter um 1% (w / v) de solução. Misturar o sal de sódio do ácido algínico, durante pelo menos 2 horas no misturador de vórtice para garantir a dissolução completa. Filtra-se a solução de alginato através de filtro de seringa de 0,2 um. Guardar a solução de alginato a 4 ° C. </li><li> Reconstituir factores de crescimento recombinantes humanas tais como fator de crescimento endotelial vascular (VEGF) ou beta - fator de crescimento neural (NGF), como recomendado pelo fabricante. </li></ol> 2. Desenho de Fibras IPC <ol><li> Misturar proteínas, factores de crescimento ou outras biomoléculas em 10-20 ul alíquota da solução de polielectrólito que tem uma carga global semelhante. Moléculas biológicas com carga negativa (por exemplo, soro albumina bovina [BSA]) líquido deve ser misturado com solução de alginato. As moléculas biológicas com carga positiva (por exemplo, VEGF) deve ser misturado comsolução de quitosano. </li><li> Coloque pequenas aliquotas (10-20 ul) de quitosana e alginato sobre uma superfície plana e estável que é coberta com parafilme. As gotas de alginato de chitosano e deve ser colocado em estreita proximidade, mas não estão em contacto uns com os outros. </li><li> Levemente mergulhe cada ponta em um par de fórceps nas gotículas de quitosana e alginato. Traga as gotas de polieletrólitos em conjunto por beliscar a pinça. Quando as gotículas entram em contacto uns com os outros, lentamente puxar a pinça na vertical para cima, para chamar a fibra IPC a partir da interface das duas gotas (Figura 1A). </li><li> Com cuidado, coloque a extremidade da fibra desenhada IPC sobre a pinça sobre um coletor, como um andaime polimérico plano aposta em um mandril rotativo (consulte a secção 3 e 4). Rodar o mandril a uma velocidade constante de 10 mm / s para permitir a formação de fibras uniforme e beadless IPC. Aumentando a velocidade de desenho das fibras IPC irá formar grânulos, que irá causar uma libertação brusca de incorporadaterminação de fibra bioquímica e prematura. 10 </li><li> Para determinar a eficiência de incorporação, recolher todos os líquidos remanescentes deixado no parafilme por diluição com 500 mL de 1X tampão fosfato salino (PBS). Medir o teor de proteína com factor de crescimento ou no resíduo através de ensaio BCA (para BSA), ELISA (para o VEGF e o NGF) ou um ensaio adequado para detectar biomolécula incorporada. </li></ol> 3. Fabricação de Composite Hidrogel Andaime de pululano-dextrano (PD) polissacarídeo e Fibras IPC <ol><li> Fabricando quadro pululano sacrificial para a coleta de fibra IPC <ol><li> Pesar pululano polissacárido e misturar com água desionizada destilada (DDI) de água para criar uma 20% (w / v) de solução aquosa. Misturar a solução de pululano O / N para assegurar a homogeneidade. </li><li> De molde 15 g de uma solução de pululano em de 10 cm de diâmetro de poliestireno de cultura de tecidos (TCPS) prato. Seca-se a solução de pululano O / N a 37 ° C. Corte os filmes de pululano em 7mm x 7 milímetros quadros quadrados. </li></ol></li><li> Preparar solução de polissacárido pululano-dextrano <ol><li> Criar uma solução a 30% dos polissacáridos de pululano e dextrano (w / v) (3: 1) em água DDI. Misturar O / N para assegurar a homogeneidade da solução de polissacárido. Adiciona-se lentamente em bicarbonato de sódio à solução de polissacárido para atingir uma concentração final de 20% (w / v). Misturar O / N para assegurar a homogeneidade da solução. Guardar a solução de polissacárido, a 4 ° C. </li></ol></li><li> Coletando fibras IPC no quadro pululano <ol><li> Apor a moldura pululano sacrificial (ponto 3.1) usando um jacaré. Furar o jacaré e estrutura pululano no final do mandril rotativo usando fita adesiva revestida de plástico. Rodar o mandril com o quadro afixada a uma velocidade constante de 10 mm / seg. O quadro de pululano pode ser afixada sobre o mandril em rotação em orientações desejadas. </li></ol></li><li> Desenhe as fibras IPC usando um par de fórceps (seção 1) e attach, a fim de as fibras desenhada IPC sobre a armação rotativa pululano. Desenhe as fibras IPC a uma velocidade constante. Ao alcançar a extremidade terminal da fibra IPC, secar o construto fibras ó-em-quadro / N à temperatura ambiente. </li><li> Incorporação fibras IPC em PD hidrogel andaime </li><li> Para reticular cada grama de solução de pululano-dextrano, adicionar 100 ul de 11% (w / v), trimetafosfato de sódio e solução aquosa de hidróxido de sódio 10M 100 ul. 7 Misture a solução a 60 rpm usando um stirplate para 1 a 2 min. Após a adição de trimetafosfato de sódio e hidróxido de sódio, a solução de polissacárido vai reticular quase imediatamente. Despeje a solução de polissacarídeo viscoso para a construção de fibras-on-frame para incorporar totalmente as fibras do IPC. Incubar as fibras de pululano-dextrano combinado IPC (IPC-PD), a 60 ° C durante 30 minutos para formar um andaimes compostos quimicamente reticulados (Figura 1B). </li><li> CUIDADO: Execute o passo 3.3.2 no exaustor e utilizar equi proteção adequadovolvimento como o ácido acético é um corrosivo e inflamável. </li><li> Para induzir a formação de poros no andaime PD-CIP, submergir todo o andaime em 20% (w / v) de ácido acético durante 20 min. </li><li> Remover os reagentes que não reagiram por lavagem andaimes PD-CIP em 1X PBS durante 5 min, agitando a 100 rpm. Repita esta etapa duas vezes. </li><li> Retire o excesso de PBS e congelar imediatamente os andaimes PD-IPC a -80 ° CO / N. Liofilizar os andaimes, pelo menos, 24 horas antes da utilização em quaisquer ensaios de libertação ou de bioatividade controladas. </li></ol> 4. Fabricação de Composite Andaime de PCL e Fibras IPC CUIDADO: Diclorometano é um material perigoso. Use a coifa e equipamentos de proteção individual ao manusear diclorometano. <ol><li> Criação de substratos PDMS puras e padronizadas <ol><li> Criar um polidimetilsiloxano intocada (PDMS) substrato elastomérico usando um pedaço de TCPS de dimensão desejada usando o processo de litografia macia. Criar pattePDMS rned substratos, usando métodos padrão suaves litográficas de poli (metacrilato de metilo) moldes com a topografia desejada. 12 </li></ol></li><li> Confecção de sacrifício PCL quadro para a recolha de fibra IPC <ol><li> Pesar PCL e dissolver em diclorometano, para se criar um 0,9% (w / v) de solução. Para cada 1 cm2 de área do substrato PDMS, gota a 500 ul de solução a 0,9% de PCL. Permitir que todo o solvente se evapore completamente diclorometano, na hotte. Repetir o processo de fundição 0,9% de PCL para engrossar a película com a espessura desejada. Remover a película de PCL seca a partir do substrato de PDMS. Criar um buraco no PCL quadro usando um perfurador de dimensões adequadas. 2 </li></ol></li><li> Coletando fibras IPC na armação PCL <ol><li> Apor a moldura PCL sacrificial com furo (de 4.2.1) em um jacaré. Furar o jacaré para o mandril rotativo usando fita adesiva revestida de plástico. Fixe a extremidade da fibra desenhada IPC para o fram PCLe antes de iniciar a rotação a uma velocidade constante de 10 mm / sec (seção 2). Após o final do estiramento de fibra IPC, secar a fibra-a-quadro constructo O / N a 4 ° C. </li></ol></li><li> Incorporação de fibra-on-frame construção em substrato PCL modelado <ol><li> Gota a 500 ul de solução a 0,9% de PCL sobre o substrato PDMS para criar uma base de PCL ou modelado como novo, como necessário. Fundido múltiplas camadas de uma solução de PCL 0,9% para se obter um andaime com a espessura desejada. Permitir que todo o solvente se evapore completamente diclorometano, na hotte. </li><li> Coloque a construção de fibra-a-quadro (secção 4.3.1) na parte superior da base de PCL. Adicionar solução de 0,9% de PCL sobre os vários tempos de fibra-a-quadro construir para obter a espessura desejada e incorporar totalmente as fibras IPC, fabricando um andaime composta IPC-PCL (Figura 1C). </li></ol></li></ol> 5. Medição da disseminação de agentes biológicos a partir Composite IPC Andaimes <ol><li> Coloque compósito PD-IPC ouAndaimes PCL-IPC e stand-alone fibras IPC separadamente em uma placa de 24 poços. </li><li> Mergulhe o andaime e stand-alone fibras IPC com 500 mL de 1X PBS. Incubar as amostras a 37 ° C. Recolhe PBS a vários pontos de tempo (libertação media) e substituir com 500 ul de PBS 1X. </li><li> Medir a quantidade de proteína ou de factor de crescimento nos meios de libertação utilizando um ensaio BCA (BSA), ELISA (VEGF e NGF) ou outro teste adequado para calcular o perfil de libertação cumulativa para a biomolécula incorporada. </li></ol> 6. Sementeira de Células no Composite IPC Andaimes para testar Bioatividade de Agentes Biológicos Lançado <ol><li> Esterilizar os scaffolds compostos PD-IPC ou PCL-IPC liofilizados utilizando luz UV na cabine de segurança biológica por pelo menos 20 min. </li><li> Utilizar técnicas padrão de cultura de células para se obter uma suspensão de células de 2 x 10 5 células em 200 ul de meio de crescimento. Semente da suspensão de células concentradas sobre os scaffolds compostos. After 20 min, top-up o volume de meio de crescimento para submergir completamente os andaimes. </li><li> Medir a atividade das células através de técnicas convencionais, tais como ensaio de actividade Alamar azul metabólica, ensaio excrescência de neurites PC12 ou imunofluorescência. </li></ol>

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The authors have nothing to disclose.

IPC fibras são formados pela interacção de dois polielectrólitos de cargas opostas. O processo utiliza a extracção do complexo a partir da interface de os polielectrólitos, facilitar um processo de auto-montagem para a formação da fibra estável. O mecanismo de formação da fibra IPC assegura que qualquer biomolécula adicionados a um polielectrólito de forma semelhante carregada pode ser incorporado durante o processo de complexação. 10,11 Inversamente, a adição de uma biomolécula para o poli...

The extracellular matrix has inherent biochemical and biophysical cues that direct cell behaviors. Mimicking this physiological three-dimensional (3D) microenvironment is a widely explored strategy for regenerative medicine and tissue engineering applications. For example, both naturally-derived and synthetic substrates have been modified with topographical cues as a means to mimic the biophysical cellular environment.1 For example, polycaprolactone (PCL) scaffolds can be easily patterned by casting on patterned PDMS substrates.2 However, most synthetic scaffolds inadequately recapitulate the controlled biochemical environment in vivo. Bulk or surface modification of synthetic materials only present biochemical cues for cell attachment but still lack temporal regulation of biochemical delivery.3 Thus, there is a need for optimal scaffolds that can mimic the temporally regulated biochemical delivery system of the extracellular matrix. 
Biochemical delivery systems such as microspheres are plagued by problems of loss of bioactivity and low incorporation efficiency due to the severity and complexity of multi-step synthesis process.4-6 Alternative methods that use a one-step fabrication and incorporation method were proven to have excellent potential to create a favorable biochemical microenvironment without the accompanying inefficiency in incorporation and loss of bioactivity. One viable solution is the use of interfacial polyelectrolyte complexation (IPC) fibers to deliver and protect biological agents. When two oppositely charged polyelectrolyte aqueous solutions are brought together, IPC fibers can be drawn out from the interface. Virtually any type of hydrophlic biomolecule in aqueous solution can be added into either the negatively- or positively-charged polyelectrolyte solution, thus facilitating the incorporation of useful biomolecules into the IPC fiber during the complexation process. Furthermore, this process only requires aqueous and ambient conditions, thereby decreasing the risk of loss of bioactivity. Using this method, active growth factors2,7 even cells8,9 have been successfully delivered. In addition, the simple method of forming IPC fibers allows molding into any shape or orientation. The stability of such fibers has been advantageous in its incorporation into both hydrophobic2 and hydrophilic polymers7 to create composite scaffolds. These composite scaffolds with IPC fibers are beneficial for creating a physiologically relevant biochemical environment while providing physical anchorage for cells. 
In this study, we show a method to incorporate IPC fibers into a hydrophilic and a hydrophobic scaffold with topography for controlled release of active biomolecules. As a proof-of-concept, we incorporate IPC fibers made from chitosan and alginate into the biocompatible, non-immunogenic and non-antigenic pullulan-dextran hydrophilic hydrogel or the biocompatible polycaprolactone hydrophobic scaffold.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pullulan&#160;</td>
			<td>Hayashibara Inc Okayama Japan</td>
			<td></td>
			<td>Molecular weight (MW) 200 kDa. This material is pharmaceutical grade pullulan used to make pullulan frames and PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma Aldrich</td>
			<td>D1037</td>
			<td>MW 500 kDa. This material is no longer being produced by Sigma Aldrich. Alternative suggested is catalog number 31392 (Sigma Aldrich). This material is used to make PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Sodium bicarbonate must be slowly added to the pullulan-dextran polysaccharide solution. Rapid addition of sodium bicarbonate will result in precipitation.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Trimetaphosphate</td>
			<td>Sigma Aldrich</td>
			<td>T5508</td>
			<td>This chemical is hygroscopic and must be stored in the dehumidifying cabinet. Aqueous solution of sodium trimetaphosphate must always be made fresh.</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>This material is hazardous and must be handled with proper protective equipment such as nitrile gloves.</td>
		</tr>
		<tr>
			<td>Chitosan</td>
			<td>Sigma Aldrich</td>
			<td>448877</td>
			<td>MW 190-310 kDa. Acetylation degree of 75 to 85%. Purification of chitosan is required to create stable IPC fibers.</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Merck</td>
			<td></td>
			<td>This can be replaced by another brand type. This material is corrosive and flammable. Protective equipment such as face shield, nitrile gloves, lab coat and shoe cover must be worn when handling this chemical in the fume hood.&#160;</td>
		</tr>
		<tr>
			<td>Alginic acid sodium salt from brown algae, low viscosity</td>
			<td>Sigma Aldrich</td>
			<td>A2158</td>
			<td>Dissolve in water overnight. Filter through sterile 0.2&#181;m syringe filter before use. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sinopharm Chemical Reagent</td>
			<td></td>
			<td>Dissolve in sterile PBS and filter using 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>This kit was used to measure BSA release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Human Recombinant Vascular Endothelial Growth Factor</td>
			<td>R&#38;D systems</td>
			<td>293-VE</td>
			<td>Dissolve growth factor in 0.2% heparin solution to a final concentration of 5 mg/ml.</td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt From Porcine</td>
			<td>Sigma Aldrich</td>
			<td>H3393</td>
			<td>This can be replaced by another brand type. Dissolve heparin salt in sterile water at a final concentration of 1% and filter through 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td>Lonza</td>
			<td>C2517A</td>
			<td>This primary cell type was used in the assay to determine VEGF bioactivity after release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Alamar blue</td>
			<td>Life Technologies</td>
			<td>DAL1025</td>
			<td>This is used to measure cell metabolic activity. Incubate Alamar blue with cells and maintain in standard cell culture conditions for 2 to 4 hours. Measure absorbance at 570 nm to determine Alamar blue percent reduction, which is correlated to the cell activity.&#160;</td>
		</tr>
		<tr>
			<td>ScanVac Coolsafe Lyophilizer</td>
			<td>Labogene</td>
			<td>7.001.200.060</td>
			<td>This is a non-programmable freeze dryer that operates at -105 to -110 &#176;C, This can be replaced by other standard lab lyophilizers.</td>
		</tr>
		<tr>
			<td>Polycaprolactone (PCL)</td>
			<td>Sigma Aldrich</td>
			<td>181609</td>
			<td>MW 65 kDa. This is no longer being manufactured by Sigma Aldrich. This can be replaced by Sigma Aldrich catalog number 704105.</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>V800151</td>
			<td>This can be replaced by another brand type. This material is hazardous and must be handled in the fume hood. Protective equipment must be worn at all times when handling this chemical.</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS; 184 Silicone Elastomer Kit)</td>
			<td>Dow Corning</td>
			<td>(240)4019862</td>
			<td>The elastomer kit comes with polymer base and crosslinker. Mixing the polymer base and crosslinker in different ratios will result in different stiffness of the PDMS.</td>
		</tr>
		<tr>
			<td>Human Recombinant Beta-Nerve Growth Factor (NGF)</td>
			<td>R&#38;D systems</td>
			<td>256-GF</td>
			<td>Reconstituted in sterile DI water to a final concentration of 100 &#181;g/ml. Aliquot and store in -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells (hMSC)</td>
			<td>Cambrex</td>
			<td></td>
			<td>This cell type was used in the assay to determine synergistic effect of NGF and nanotopography.</td>
		</tr>
		<tr>
			<td>Rat PC12 Pheochromocytoma Cells&#160;</td>
			<td>ATCC</td>
			<td></td>
			<td>This cell type was used in the neurite outgrowth assay to determine bioactivity of NGF. After exposure to release media with NGF, measure number of cells with neurite extensions and normalize to total number of cells.</td>
		</tr>
		<tr>
			<td>Grade 93 filter paper</td>
			<td>Whatman</td>
			<td>Z699675</td>
			<td>This is used for the purification of chitosan after its precipitation with sodium hydroxide at pH 7.</td>
		</tr>
		<tr>
			<td>Swing bucket centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>To be used during the purification of chitosan using 1200 x g speed.&#160;</td>
		</tr>
		<tr>
			<td>Motor with mandrel rotating at constant speed</td>
			<td>Rhymebus</td>
			<td>RM5E</td>
			<td>The motor is used for the fabrication of IPC fibers on pullulan or PCL frame.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>FirstBase</td>
			<td></td>
			<td>Sterilize through filtration (0.2 &#181;m filter) and autoclave.&#160;</td>
		</tr>
		<tr>
			<td>10-mm diameter Tissue Culture Polystyrene Dish (TCPS)</td>
			<td>Greiner</td>
			<td></td>
			<td>The TCPS dish is used for casting of pullulan frame.&#160;</td>
		</tr>
		<tr>
			<td>Human VEGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DVE00</td>
			<td>The ELISA kit is used for detection of VEGF in the release medium.</td>
		</tr>
		<tr>
			<td>Human NGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY256</td>
			<td>&#160;The ELISA kit is used for detection of NGF in the release medium.</td>
		</tr>
		<tr>
			<td>Plastic Coated Adhesive Tape</td>
			<td>Bel-Art</td>
			<td>9040336</td>
			<td>The adhesive tape is used to securely stick the alligator clip to the rotating mandrel</td>
		</tr>
	</tbody>
</table>

composite scaffolds of interfacial polyelectrolyte fibers for temporally controlled release of biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Neste artigo, buscou-se criar scaffolds compósitos com fibras de IPC para a libertação controlada de várias biomoléculas. Características das biomoléculas utilizadas neste estudo encontram-se na Tabela 1. Fibras de IPC foram incorporados primeiramente em um hidrogel hidrofílico PD para criar um andaime composta PD-CIP (Figura 1B). Molécula modelo BSA foi testado pela primeira vez para determinar a viabilidade da utilização de um andaime composto para liberação controlada de...

Watch this Scientific Journal Video about Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules at JoVE.com

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Andaimes de mistura de fibras Interfacial polieletrólito para lançamento Temporalmente controlada de biomoléculas

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

composite-scaffolds-interfacial-polyelectrolyte-fibers-for-temporally

Research

JoVE Journal

Bioengineering

8.3K visualizações.  National University of Singapore. Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Vídeo: Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

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

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparação de fibrina Andaimes 3D para aplicações de cultura de células-tronco

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sem contato, Monitoramento Label livre de células e matriz extracelular utilizando Espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

Preparação de Membranas de Luz respondem por um enxerto de superfície combinada e Processo Postmodification

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Alternando microgéis de gelatina híbrido Campo-Responsive magnética para controlada de fármacos de libertação

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Geração de células de camundongo Airway epiteliais derivados de ESC Usando descelularizado Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

Complexo polielectrólito de ligação à heparina de entrega osteogénica Factor de Crescimento Domínio

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Preparação de Matriz Extracelular fibras de proteína de Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Poli (Magnetic and Thermal-sensitive Poly)<em&gt; N</em&gt; Microgels com base em isopropilacrilamida) para liberação controlada com disparo magnético

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Fabricação de matriz de cartilagem-derivado Decellularized andaimes

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...