The authors would like to acknowledge Stanford Department of Orthopaedic Surgery and Stanford Coulter Translational Seed Grant for funding. J.H.L. would like to thank National Science Foundation Graduate Fellowship and DARE Doctoral Fellowship for support.

Todos os experimentos foram realizados de acordo com as orientações dos sujeitos Stanford University Humanos e aprovado protocolo Institutional Review Board. 1. Articular condrócitos Isolamento <ol><li> Obter cartilagem a partir de tecido descartado durante artroplastia total do joelho e dissecar como descrito anteriormente 8. </li><li> Visualmente seleccionar os côndilos femorais mediais ou laterais de amostras de cartilagem de superfícies lisas e fazer incisão na superfície da cartilagem por meio de um bisturi de forma a remover as biópsias 4-5 mm de cartilagem sem afectar o osso subcondral. </li><li> Isolar condrócitos a partir da matriz extracelular por dissociação enzimática do tecido com tratamento / N O com colagenase purificados. Use proporção 1: 1 de colagenase II (125 U / ml) e a colagenase IV (160 U / mL) em meio de crescimento de condrócitos a 37 ° C. </li><li> No dia seguinte, filtra-se o tecido digerido contendo as células dissociadas através de uma 70mm tamanho dos poros da malha de nylon. Lavar duas vezes com 20 ml de DMEM / F12 e centrifugar a 600 xg durante 5 min. </li><li> Placa os condrócitos isoladas a uma densidade de 1 x 10 6 células / 60 mm prato de cultura a seguir re-suspensão em meio DMEM / F12 suplementado com 10% de Soro Fetal Bovino (FBS), 25 ug / ascorbato mL, 2 mM de L-glutamina, antimicrobianos (100 U / ml de penicilina, 100 ug / ml de estreptomicina, e 0,25 ug / ml de fungizona). Manter as placas a 37 ° C e manter as culturas de condrócitos primários como uma monocamada de alta densidade antes da encapsulação em hidrogéis biomiméticos. </li></ol> 2. Biomimético Hidrogel Fabrication NOTA: Este método permite a síntese de um hidrogel biomimética contendo poli (etileno-glicol diacrilato) (PEGDA, PM 5000 g / mol), sulfato de condroitina-metacrilato (CS-MA) em DPBS. A adição de foto-iniciador de reticulação permite fotoactivado. A composição de hidrogel final contém 7% pesart / volume (w / v) de PEGDA, 3% w / v de CS-MA, e 0,05% w / v do foto-iniciador. <ol><li> Sintetizar CS-MA por funcionalização polímero CS com grupos de metacrilato. <ol><li> Preparar uma solução tamponada de 50 mM de ácido 2-morpholinoethanesulfonic (MES) 0,5 M e cloreto de sódio (NaCl) por dissolução de 1,952 g de MES e 5,84 g de NaCl em 200 ml de água desionizada (DIH 2 O). Dissolve-se 5 g de sal de sulfato de condroitina de sódio na solução tamponada. </li><li> Adicionar 0,532 g (4,62 mmol) de N-hidroxissuccinimida (NHS) e 1,771 g (9,24 mmol) de 1-etil-3- (3-dimetilaminopropil) -carbodiimida (EDC) (proporção molar de NHS: EDC = 1: 2) para a solução e agita-se durante 5 min. </li><li> Adicionar 0,765 g (4,62 mmol) de metacrilato de 2-aminoetilo (AEMA) (proporção molar de NHS: EDC: AEMA = 1: 2: 1) à solução e manter a reacção à temperatura ambiente durante 24 h. </li><li> Purifica-se a mistura através de diálise contra água desionizada (DIH 2 O) durante 4 dias, utilizando tubagem de diálise (MWCO de 12-14 kDa). </li><li> Filtra-se a purified solução através de um filtro de 0,22 um e coloque os tubos abertos contendo a solução no exsicador de vácuo e S / N. Assegurar que todos os tubos são protegidas da luz. Armazenar o polímero dissolvido à temperatura de -20 ° C ao abrigo da luz e humidade. </li></ol></li></ol> Encapsulation 3. celular <ol><li> Autoclave o filme e cilíndricas hastes de PCR (para perfurar os hidrogéis 3D de células-laden) e esterilizar o molde gel cilíndrico feito por encomenda por submersão em 0,2 m filtrada 70% de etanol em cultura de tecidos capô sob luz UV. </li><li> Fecha-se o fundo do molde de gel com película de PCR autoclavado sem bolhas de ar ou aberturas para evitar a fuga. Mantê-lo em um estéril placa de 150 mm. </li><li> O dia antes da encapsulação de células em hidrogel biomimética, dissociar a condrócitos de monocamada de alta densidade com tratamento / N O em colagenase II e solução de colagenase IV. Use 125 U / ml para a colagenase II e 160 U / ml de colagenase para cada VI em meios de crescimento de condrócitosa 37 ° C. </li><li> Tomar um tubo de 50 ml e adicionam-se 5% em peso / volume (w / v) (diacrilato de etileno-glicol) poli (PEGDA, PM = 5.000 g / mol), 3% w / v de sulfato de condroitina-metacrilato (CS-MA), e 0,05% w / v fotoiniciador em DPBS. Vortex do tubo para misturar a solução e mantê-lo de lado. Proteger o tubo de luz. </li><li> Recolher as células dissociadas num tubo Falcon de 50 ml, contar e centrifugar a 460 xg durante 5 min. Aspirar todo o material da tubo de células e adicionar o material de gel misturado acima para re-suspender as células a uma densidade de 15 x 10 6 células / ml. Misturá-lo 30 vezes completamente, evitando a formação de bolhas de ar. </li><li> Pipetar 72 ul da suspensão de células de hidrogel ou hidrogel sozinho para dentro do molde de gel cilíndrico feito por encomenda e induzir a gelificação por exposição à luz UV (365 nm de comprimento de onda) a 3 mW / m 2 durante 5 min. Prepare alguma solução de hidrogel sem conteúdo celular. Use essas construções como controlos negativos para o futuro de testes bioquímicos e mecânica. Measure a intensidade da luz UV com um medidor de intensidade da radiação UV. Para ajustar a intensidade, alterar a distância entre a fonte de luz UV e o andaime hidrogel durante a gelificação. Observação: Uma vez que o hidrogel contém CS, a contribuição acelular é subtraído do conteúdo de GAG ​​bioquímica dos hidrogéis de células carregadas para representar apenas o GAG celular. </li><li> Use um bisturi para cortar o filme para fácil peel off e com cuidado remova-o. Com a ajuda das hastes cilíndricas, empurrar para fora do gel para uma placa de 6 poços com 5 ml de DPBS estéril para lavar gel não polimerizado e células soltas. </li><li> Cultura os hidrogéis de células carregadas em placas de 24 poços contendo 1,5 mL de meio de crescimento de condrócitos em cada poço. Usar uma espátula descartável para transferir os hidrogéis lavou no bem. </li><li> Avaliar a viabilidade celular por mortos vivem coloração 24 horas pós-encapsulamento usando um kit de viabilidade / citotoxicidade. Cultura os hidrogéis de células-laden e os hidrogéis vazios para 3-6 semanas mudando para gro de condrócitos frescoswth mídia a cada 2 dias antes da colheita para as análises. </li></ol> 4. RNA Extração e Análise de Expressão Gênica <ol><li> Aspirar cuidadosamente a mídia e colocar estéril PBS 1X na célula-laden, bem como os hidrogéis de controle vazios. </li><li> Com a ajuda de uma espátula estéril, transferir cada um hidrogel a um tubo eppendorf de 1,5 ml limpo e adicionar 250 ul de Tri-reagente a cada tubo. Siga as instruções do fabricante para isolar RNA. </li><li> Após o isolamento de RNA e quantificação utilização de 1 ug de cada amostra que e efectuar uma transcrição invertida em ADNc utilizando o High Capacity cDNA Kit de Transcrição Reversa. </li><li> Para PCR quantitativo usar TaqMan Gene Expression Arrays para exame da expressão de seus genes de interesse. Normalizar os níveis de expressão do gene de GAPDH internamente. </li><li> Para as condições de PCR incluem a 2 min de incubação a 50 ° C para inactivar amplicões anteriores com glicosilase de uracilo de ADN, seguido por um 1060; min de incubação a 95 ° C para activar a polimerase de Taq. Em seguida, executar ciclos de PCR quarenta, que consistem de 15 seg a 95 ° C e 1 min a 60 ° C. </li></ol> 5. As análises bioquímicas <ol><li> Para cada estruturas celulares de hidrogel quantificar DNA e glicosaminoglicanos sulfatados (sGAG) produção como acompanhamento. </li><li> Aspirar cuidadosamente a mídia e colocar estéril PBS 1X na célula-laden, bem como os hidrogéis de controle vazios. Pesar um tubo vazio e colocar o hidrogel dentro após a secagem-lo em papel de seda e registrar o peso ou peso úmido dos hidrogéis. </li><li> Congelar cada hidrogel à temperatura de -20 ° C em um tubo Eppendorf de 1,5 ml separado para a digestão enzimática e depois usar-se uma alíquota do produto digerido a executar ensaio de Picogreen (para o DNA) e dimetil-metileno ensaio de azul (por GAG). </li><li> Preparar a solução de papainase por digestão enzimática dos hidrogéis. <ol><li> Primeiro prepare o tampão PBE por pesando 7,1 g de sódio phosphate dibico (Na 2 HPO 4) e 1,6 g do ácido etilenodiaminotetracético, sal dissódico (EDTA-Na2). Dissolver em 500 ml de dH 2 O, ajustar o pH para 6,5 e a solução purificada filtrar através de um filtro de 0,22 mm. </li><li> Dissolve-se 0,035 g de L-cisteína em 20 ml de tampão de PBE. Filtra-se a solução e 100 ul de enzima papaína estéril. </li></ol></li><li> Quando estiver pronto para realizar os ensaios, levar os tubos de -20 ° C e adicionar 300 ul da solução de papaína pronto para os hidrogéis congelados. Esmagar o gel com um almofariz e pilão e misture bem com o pilão motor. </li><li> Levar o volume para 500 ul com solução de papaína adicional. Realizar o mesmo procedimento para os geles de controlo. Incubar a 60 ° C durante 16 horas 3,9. A solução contendo o hidrogel pode digerido por conseguinte, utilizados para quantificação de ADN e de GAG. Medir o teor de ADN, utilizando o ensaio de Picogreen com fago lambda de ADN como padrão seguinte PR do fabricanteotocol. </li><li> Quantificar o conteúdo sulfatada-GAG utilizando o azul 1,9-dimetilmetileno (DMMB) ensaio de ligação de corante com sulfato de condroitina tubarão como padrão 10. Determinar o teor de GAG ​​dividindo-se a quantidade de GAG ​​para o peso húmido correspondente. </li></ol> 6. Ensaios Mecânicos <ol><li> Realizar ensaios de compressão simples, utilizando um Instron 5944 sistema de testes equipada com uma célula de carga de 10 N. <ol><li> Depois dos dias desejados de cultura in vitro, execute o teste de compressão no cadafalso célula-hidrogel e os hidrogéis de controle acelular. </li><li> Submergir as amostras em um banho de PBS à temperatura ambiente e comprimir a uma taxa de 1% de deformação / seg para uma tensão máxima de 15% 11,12 desde o esforço fisiológico experimentado por tecido de cartilagem sob condições de carga tem sido relatada como sendo de 10-20% 13,14. </li></ol></li><li> Criar estresse vs. curvas de tensão e ajuste de curva usando uma equação polinomial de terceira ordem. Determinar os comprmódulo tangente essive partir da curva de ajuste equação em valores de deformação de 15%. </li></ol>

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The authors have nothing to disclose.

Conforme relatado no presente protocolo, os hidrogéis 3D são capazes de manter condrócitos fenótipo na cultura, evitando o processo de desdiferenciação celular em células fibrocartilagem normalmente encontrados com culturas em monocamada 15. Além disso, as culturas a longo prazo do construto hidrogel chondrocytes- revelou um ambiente favorável que mantém as características intrínsecas de células associadas com a idade e a doença. A utilização de um hidrogel biomimética 3D tem várias vantagens. Em primeiro lugar, a inclusão de sulfato de condroitina (CS), um componente principal encontrado na cartilagem articular, permitir que as células de degradar a matriz de hidrogel através da secreção de condroitinase e deitou-se cartilagem recém-sintetizado extra-celular da matriz 5, 16. Além disso, o CS foi mostrado que têm propriedades anti-inflamatórias nas articulações artríticas. O hidrogel biomimética também pode ser utilizado como um material de andaimes para entrega em células de reparação de cartilagem, e podem ser quimicamente modificadospara facilitar uma melhor integração 17,18 tecido-biomaterial. A utilização de hidrogeles de PEG-CS permite culturas a longo prazo de condrócitos e a avaliação das propriedades bioquímicas e mecânicas. Aqui mostramos como esta plataforma pode ser útil para as análises comparativas de diferentes fontes de condrócitos diferenciados, a fim de definir o tipo celular ideal para a engenharia de cartilagem. Curiosamente, condrócitos encapsulados em hidrogéis permanecer viável e proliferam de acordo com as suas capacidades intrínsecas. Os suportes composição de hidrogel, na verdade, o crescimento de jovens e adultos condrócitos saudáveis, como mostrado na Figura 2. A composição e estrutura dos hidrogeles descritos também promove a formação de tecido de cartilagem, como indicado pela deposição de uma matriz extracelular funcional avaliada por glicosaminoglicano (GAG ) quantificação. Uma vantagem adicional é que as construções de condrócitos-hidrogelpode ser avaliada para as propriedades mecânicas do tecido de cartilagem recém-formado. Note-se que o ensaio de compressão simples deve ser executada no hidrogel acelular para comparação. Os hidrogeles, de facto, tem uma rigidez intrínseca devido à rigidez das porções CS. Estirpe compressão simples de 5-20% (a uma velocidade de deformação de 1% / s) podem ser aplicados para o teste mecânico de tecido de cartilagem 11,12 desde o esforço fisiológico experimentado por tecido de cartilagem sob condições de carga tem sido relatada como sendo 10-20 % 13,14. A resposta de ambos célula de carga e acelulares hidrogéis ao teste mecânico foi avaliada no ponto final da cultura. No exemplo descrito acima, observou-se uma rigidez comparável das construções contendo adulto e juvenil condrócitos em contraste com a rigidez mais baixa das construções contendo condrócitos OA. Tais propriedades mecânicas da construção de células-hidrogel permitem a avaliação das propriedades funcionais dotecido formado dando uma análise aprofundada da capacidade maturação celular. Em conclusão, a utilização dos hidrogeles biomiméticos 3D para estudar o potencial de diferente população de condrócitos para gerar tecido de cartilagem pode ser amplamente aplicada. Além dos estudos in vitro descrito aqui, o transplante in vivo das construções de células-carga pode ser concebido para estudar a maturação de células e o potencial regenerador no contexto fisiológico. Outras modificações da plataforma de hidrogel com factores adicionais biomiméticos também pode ser prevista para optimizar a proliferação de condrócitos e maturação.

With its limited self-repair potential, human articular cartilage undergoes frequent irreversible damages. Extensive efforts are currently focused on the development of efficient cell-based approaches for treatment of articular cartilage injuries. The success of these cell-based therapies is highly dependent on the selection of an optimal cell source and the maintenance of its regenerative potential. Chondrocytes are a common cell source for cartilage repair, but they are limited in supply and can de-differentiate during in vitro expansion in 2D monolayer culture thereby limiting their generation of hyaline cartilage 1. 
The aim of this protocol is to establish a 3-dimensional hydrogel platform for an in vitro comparative study of human chondrocytes from different ages and disease state. Unlike conventional two-dimensional (2D) culture, three-dimensional (3D) hydrogels allow chondrocytes to maintain their morphology and phenotype and provides a physiologically relevant environment enabling chondrocytes to produce cartilage tissue 2,3. In addition to providing a 3D physical structure for chondrocyte culture, hydrogels mimic the function of native cartilage extracellular matrix (ECM). Specifically, the inclusion of chondroitin sulfate methacrylate provides a potential reservoir for secreted paracrine factors 4 and enables cell-mediated degradation and matrix turnover 5. Although many 3D hydrogel culture systems have been utilized widely in various studies including agarose and alginate gels, we have used a biomimetic 3D culture system that has some distinct advantages for chondrocyte culture. Chondroitin sulfate (CS) is an abundant component in articular cartilage and the PEG-CS hydrogels have been shown to maintain and even enhance chondrogenic phenotype and facilitate cell-mediated matrix degradation and turnover 2,5. In addition, the mechanical properties of the hydrogel scaffold can be easily modulated by changing concentration of PEG and hence can be utilized to further enhance the regeneration potential of chondrocytes or a related cell type 6,7. PEG/CSMA is also biocompatible and hence has the potential for a direct clinical application in cartilage defects for example. The limitation for this system is its complexity and the use of photopolymerization that can potentially affect cell viability as compared to simpler systems like agarose, however the advantages for the chondrocyte culture outweigh the potential limitations.
The 3D hydrogel culture is compatible with conventional assay for evaluation of cell phenotype (gene expression, protein immunostaining) and functional outcome (quantification of cartilage matrix production, mechanical testing). This favorable 3D environment was tested to compare the tissue regeneration potential of human chondrocytes from three different aged populations in long-term 3D cultures. 
The outcomes were evaluated via both phenotypic and functional assays. Juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogel culture. After 3 and 6 weeks, chondrogenic gene expression was upregulated in juvenile and adult chondrocytes but was downregulated in OA chondrocytes. Deposition of cartilage tissue components including aggrecan, type II collagen, and glycosaminoglycan (GAG) was high for juvenile and adult chondrocytes but not for OA chondrocytes. The compressive moduli of the resulting cartilage constructs also exhibited similar trends. In conclusion, both juvenile and adult chondrocytes exhibited chondrogenic and cartilage matrix disposition up to 6 weeks of 3D culture in hydrogels. In contrast, osteoarthritic chondrocytes revealed a loss of cartilage phenotype and minimal ability to generate robust cartilage.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>juvenile chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System )</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>adult chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System)</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(ethylene glycol diacrylate)</td>
			<td>Laysan Bio</td>
			<td>ACRL-PEG-ACRL-1000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>2-morpholinoethanesulfonic acid</td>
			<td>Sigma</td>
			<td>M5287</td>
			<td></td>
		</tr>
		<tr>
			<td>photoinitiator</td>
			<td>Irgacure&#160;</td>
			<td align="right">2959</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride&#160;</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>chondroitin sulfate sodium salt&#160;</td>
			<td>Sigma</td>
			<td>C9819</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide&#160;</td>
			<td>Sigma</td>
			<td align="right">130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride</td>
			<td>Sigma</td>
			<td>E1769</td>
			<td></td>
		</tr>
		<tr>
			<td>2-aminoethyl methacrylate</td>
			<td>Sigma</td>
			<td align="right">516155</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Spectrum Laboratories&#160;</td>
			<td align="right">132700</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 2</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 4</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004189</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>HyClone, Thermo Scientific&#160;</td>
			<td>SH3002301&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>live/dead assay</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/L3224">L3224</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Tri reagent</td>
			<td>Life Technologies</td>
			<td>AM9738</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT&#8482; PicoGreen&#174; dsDNA Assay Kit</td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/P11496">P11496</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic&#160;</td>
			<td>Sigma</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt&#160;</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C1276</td>
			<td></td>
		</tr>
		<tr>
			<td>1,9-dimethylmethylene blue&#160;</td>
			<td>Sigma</td>
			<td align="right">341088</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV light equipment - XX-15LW Bench Lamp, 365nm</td>
			<td>UVP</td>
			<td>95-0042-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Instron 5944 testing system&#160;</td>
			<td>Instron Corporation</td>
			<td>E5940</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Hidrogéis bioactivos contendo PEG e porções CS (Figura 1) representam uma plataforma ideal para a cultura e maturação de condrócitos articulares humanos 2,3,5,7. Os condrócitos a partir de diferentes idades e estados de doença podem ser cultivadas com o método descrito e analisado para o seu fenótipo, a expressão do gene e propriedades bioquímicas e mecânicas do tecido de cartilagem gerado. Três amostras de condrócitos, juvenile- 6 meses, adulto-34 anos e 74 anos osteoarthritic-, foram colhidos após 3 semanas de cultura em hidrogéis biomiméticos 3D. Análises de condrócitos normais, tanto adulto e juvenil expressão de genes, apresentaram um aumento na expressão dos genes de condrócitos COL2A1 e COL6A1. Pelo contrário, os condrócitos doentes apresentaram uma diminuição dramática no COL2A1 enquanto se mantinha a expressão de COL6A1 (Figura 2) mostra uma perda de fenótipo condrogénica apesar de ser cultivadas num ambiente biomimética favorável. Os hidrogeles de PEG-CS também servir como um ambiente dinâmico para os condrócitos a proliferar, digerir a matriz pré-existente de PEG e CS e depositar sua matriz pericelular e extracelular, os principais componentes do tecido de cartilagem madura. Tendo em conta estas qualidades, a expansão de condrócitos após 3 semanas de cultura in vitro pode ser estimada pela quantificação de ADN com o corante Picogreen. A análise comparativa dos três grupos de células demonstram que a densidade de populações de células juvenis e adultas foi inalterada enquanto os condrócitos OA exibiram uma diminuição dramática em comparação com o dia 1 de cultura. A perda de teor de ADN foi também observado por OA mas não outros condrócitos sugerindo a possibilidade de a morte das células durante a cultura a longo prazo (Figura 3). A matriz segregada foi quantificada como teor de GAG ​​sulfatado-por ensaio de ligação de corante DMMB na fase de ponto final após 3 semanas de cultura. MORDAÇA conteúdo é normalizada pelo peso úmido do hidrogel como recorDED antes da digestão enzimática e da contribuição acelular é subtraído. Como mostrado na Figura 4, os condrócitos depositada uma quantidade significativa de GAG semelhante durante 3 semanas de cultura em hidrogéis 3D. A presença de uma estrutura de suporte físico permite a avaliação de propriedades biomecânicas das amostras não confinada por meio de ensaios de compressão 2,3. Embora os hidrogéis acelulares submetido a um decréscimo na módulos de compressão, as construções de células-mantida em carga os módulos de compressão ao fim de 3 semanas em cultura (Figura 4). O fenotípica, análises bioquímicas e biomecânicas aqui descritas são, portanto, úteis para avaliar e compreender o potencial de diferentes populações de condrócitos para a cartilagem de engenharia. <img alt="figura 1" src="/files/ftp_upload/53085/53085fig1.jpg" /> Figura 1. PEG-CS hidrogéis biomiméticos para condrócitos cul 3Dtura. Os condrócitos são ressuspensas numa mistura que contém o poli (etileno glicol diacrilato) (PEGDA) e sulfato de condroitina-metacrilato (CS-MA) e fundido para dentro do molde de gel cilíndrico feito por encomenda. Após a exposição UV, solidificou géis são recolhidos a partir dos moldes e viabilidade celular é avaliada por coloração morto ao vivo 24 horas pós-encapsulamento. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53085/53085fig2.jpg" /> Figura 2. Expressão de genes condrogênicos em condrócitos humanos cultivados em hidrogéis biomiméticos 3D. Quantitativa de expressão do gene COL2A1 marcadores da cartilagem e COL6A1 por juvenis, adultos e OA condrócitos após 3 semanas de cultura em hidrogéis biomiméticos 3D. Os valores são normalizados para o nível expressão gênica no dia 1. Erro bars representam a média ± SD. * p &lt;0,05, conforme determinado por um teste t de Student bicaudal. Modificado de Smeriglio et al. 2 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53085/53085fig3.jpg" /> Figura 3. teor de ADN em condrócitos humanos cultivados em hidrogéis biomiméticos 3D. Quantificação por ensaio de ADN Picogreen em juvenis, adultos e OA condrócitos após 3 semanas de cultura em hidrogéis biomiméticos 3D. Os valores são normalizados para nível DNA no dia 1. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;br /&gt; Figura 4. GAG medição de conteúdo e teste de compressão simples de condrócitos humanos no dia 1 e depois de 3 semanas de cultura em hidrogéis biomiméticos 3D. (A) GAG quantificação por ensaio DMMB em condrócitos no dia 1 e depois de 3 semanas de cultura em hidrogéis biomiméticos 3D. Os valores são normalizados para molhar peso (ww) e expressos como ug / g. (B) módulo de compressão (kPa) de acelular e celulares carregados de hidrogéis no dia 1 e depois de 3 semanas de cultura em hidrogéis biomiméticos 3D. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D Hidrogel Andaimes para articular de condrócitos Cultura e Geração de cartilagem

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

3d-hydrogel-scaffolds-for-articular-chondrocyte-culture-cartilage

Research

JoVE Journal

Bioengineering

19.8K visualizações.  Stanford University. Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes. 

Vídeo: 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Uma simples gota de suspensão Protocolo Cultura de Células para a geração de esferóides 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

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 ...

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 ...

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 ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Um Experimental e Finite Element protocolo para investigar o transporte de solutos neutras e carregadas em toda a cartilagem articular

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D Agregação Stem Magnetic celular e biorreator Maturação para regeneração da cartilagem

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

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 ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Visualização em tempo real e análise do condrócito prejuízo devido ao carregamento mecânico em explantes de cartilagem murino totalmente intacta

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

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 ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D impresso celulose porosa nanocompósitos Hydrogel andaimes

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...