Name: 3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation
Uploaded: 2015-10-07T14:00:00.000+00:00
Duration: 12 min 37 s
Description: Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

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><tbody><tr><td>juvenile chondrocytes (Clonetics Normal Human Chondrocyte Cell System )</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>adult chondrocytes (Clonetics 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&amp;nbsp;</td><td>2959</td><td></td></tr><tr><td>sodium chloride&amp;nbsp;</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>chondroitin sulfate sodium salt&amp;nbsp;</td><td>Sigma</td><td>C9819</td><td></td></tr><tr><td>N-hydroxysuccinimide&amp;nbsp;</td><td>Sigma</td><td>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>516155</td><td></td></tr><tr><td>dialysis tubing</td><td>Spectrum Laboratories&amp;nbsp;</td><td>132700</td><td></td></tr><tr><td>Collagenase 2</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004177</td><td></td></tr><tr><td>Collagenase 4</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004189</td><td></td></tr><tr><td>DMEM/F12 media</td><td>HyClone, Thermo Scientific&amp;nbsp;</td><td>SH3002301&amp;nbsp;</td><td></td></tr><tr><td>live/dead assay</td><td>Life Technologies</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/L3224&quot;&gt;L3224&lt;/a&gt;</td><td></td></tr><tr><td>Tri reagent</td><td>Life Technologies</td><td>AM9738</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Invitrogen</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/P11496&quot;&gt;P11496&lt;/a&gt;</td><td></td></tr><tr><td>Sodium phosphate dibasic&amp;nbsp;</td><td>Sigma</td><td>S3264</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>341088</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>UV light equipment - XX-15LW Bench Lamp, 365 nm</td><td>UVP</td><td>95-0042-07</td><td></td></tr><tr><td>Instron 5944 testing system&amp;nbsp;</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

20.0K 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

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

Preparação do hidrogel injetável à base de quitosana e sua aplicação em cultura de células 3D

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Química

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Cartilagem Human Tissue Fabricação Usando tridimensional Inkjet Tecnologia de Impressão

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Bioengenharia

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Em camadas de alginato Constrói: Uma Plataforma para Co-cultura de populações de células heterogéneas

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

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

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

Cultivo de células de mamíferos em andaimes tridimensionais do Peptide

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

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Um Sistema de 3D para condrócitos Cultivar Humanos Articular no líquido sinovial

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Biologia

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

A estimulação mecânica de condrócitos-agarose Hidrogéis

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

Criação de uma articulação do joelho em um chip para modelagem de doenças articulares e testes de medicamentos

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Síntese de Hidrogéis de Matriz Extracelular de Cartilagem Descelularizada

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Geração de esferoides a partir de vários condrócitos usando condições de baixa aderência sob gravidade e mini-biorreatores caseiros

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...