Gli autori vorrebbero riconoscere QuinXell Technologies e CellD, in particolare Lothar Grannemann e Dominique Ghozlan per il loro aiuto con il bioreattore. Ringraziamo Catherine Le Visage, che ci ha fornito i pullulano / ponteggi polisaccaride destrano. Questo lavoro è stato sostenuto dall&#39;Unione Europea (progetto ERC-2014-Cog Matisse 648.779) e dalla AgenceNationalede la Recherche (ANR), la Francia (progetto MagStem ANR-11 SVSE5).

1. la costruzione dei dispositivi magnetici NOTA: I dispositivi utilizzati per semina delle cellule variano a seconda dell&#39;applicazione (Figura 1). A formare aggregati, il numero delle cellule è limitato a 2,5 × 10 5 / aggregato, così le punte magnetiche devono essere molto sottili (750 micron di diametro). Seminare i ponteggi 1,8 centimetri 2/7 mm di spessore, i magneti devono essere più grandi (3 mm di diametro) e garantiranno migrazione cellulare attraverso i pori del ponteggio. <ol><li> Costruzione di un dispositivo a micro-magneti per la formazione di aggregati (Figura 1A) <ol><li> Rendere microfori con un trapano 0,8 mm mediante piastre di alluminio (3 cm di diametro e spessore 6 mm). </li><li> Inserire una punta magnetica (750 micron di diametro) in ogni foro della piastra. </li><li> Posizionare questo disco sopra un magnete al neodimio permanente, che garantisce la magnetizzazione di saturazione. </li></ol></li><li> Costruzione di un dispositivo per ponteggio semina ( <strong&gt; Figura 1B) <ol><li> Tagliare polistirolo rigido in 2.4 cm 2 piazze. </li><li> Inserire 9 piccoli magneti (3 mm di diametro, 6 mm di lunghezza) equidistanti su una superficie di 1,6 cm 2. </li><li> Posizionare il dispositivo nel corso di un magnete al neodimio permanente. </li></ol></li></ol> 2. Stem Cell Labeling NOTA: Le cellule staminali sono state marcate con 0,1 mM nanoparticelle magnetiche per 30 min (2,6 ± 0,2 pg ferro / cella) a formare aggregati, mentre erano etichettati con 0,2 mM nanoparticelle magnetiche per 30 min (5 ± 0,4 pg ferro / cella) per seminare impalcature. Queste concentrazioni di nanoparticelle e tempi di incubazione sono stati utilizzati in precedenza e pubblicato per MSC e altre cellule 18, 19, ed è stato determinato che le nanoparticelle influenzato né vitalità cellulare né capacità di differenziazione MSC. La massa ferro incorporata dalle cellule staminali è stato misurato mediante single-cell magnetophoresis 19, 20. <ol><li> Cellule di coltura umane staminali mesenchimali (MSC) nella massima terreno di crescita delle cellule staminali mesenchimali (MSCGM) a 37 ° C e 5% di CO 2 fino vicino alla confluenza (~ 90%). </li><li> Preparare la soluzione di etichettatura magnetico miscelando 0,1 o 0,2 mM maghemite citrato rivestite con ossido di ferro (γFe 2 O 3; nucleo: 8 nm di diametro) in terreno 5A di McCoy privo di siero modificata a Roswell Park Memorial Institute (RMPI) senza glutammina e contenente 5 mM citrato di sodio. </li><li> Eliminare il mezzo, lavare le cellule con mezzo RPMI privo di siero senza glutammina, e aggiungere 10 ml di soluzione di ferro ossido di nanoparticelle a 150 cm 2 pallone di coltura, il volume minimo richiesto per coprire tutte le celle. </li><li> Incubare per 30 minuti a 37 ° C e 5% di CO 2 e quindi eliminare la soluzione di nanoparticelle. Sciacquare per 5 minuti con mezzo RPMI privo di siero senza glutammina per internalizzare il nanoparticles ancora attaccato alla membrana plasmatica. </li><li> Eliminare il medium RPMI e aggiungere 25 ml di completo media MSCGM per bombola. Incubare per una notte a 37 ° C e 5% CO 2. </li></ol> 3. semina delle cellule magnetiche <ol><li> Appena preparare il mezzo condrogenica usando Dulbecco Modified Eagle Medium (DMEM) high glucose con L-glutammina aggiungendo 50 mM di acido L-ascorbico 2-fosfato, 0,1 pM desametasone, 1 mM sodio piruvato, 0,35 mM L-prolina, 1% cultura universale supplemento contenente insulina, transferrina umana e acido selenioso (ITS-Premix), e 10 ng / mL di crescita trasformante fattore-beta 3 (TGF-β3). </li><li> Staccare le cellule magnetiche utilizzando 8 ml di 0,05% tripsina-EDTA a 150 cm-2 pallone di coltura e centrifugare le cellule dissociate a 260 xg per 5 min. Aspirare il terreno e contare le cellule risospese. </li><li> Posizionare una coltura cellulare con fondo piatto Petri (35 mm) al di sopra di entrambi i dispositivi magnetici. </li><li> per magnetically formare aggregati, aggiungere 3 ml di terreno condrogenica alla piastra di Petri e delicatamente depositare il minor volume possibile (non più di 8 mL) contenente 2,5 x 10 5 cellule marcate per aggregato (fino a 16 aggregati può essere depositato). Lasciare la piastra di Petri per 20-30 min senza spostarlo, permettendo di formare sferoidi, e quindi inserire il dispositivo completo, compresa la piastra di Petri contenente 16 aggregati, in incubatore a 37 ° C e 5% CO 2. </li><li> aggregati controllo forma seguendo lo stesso protocollo e sostituire il mezzo completo con il mezzo condrogenica senza TGF-β3. <ol><li> Per generare l&#39;aggregato costrutto 3D, mettere 2 aggregati in contatto il giorno 8 per formare 8 doppiette ed avviare la fusione. Il giorno 11, unire 2 doppiette per formare 4 quartine. Infine, fondere le 4 quadruplets il giorno 15 per ottenere la struttura finale. </li><li> Allo stesso tempo, formare aggregati centrifugando 2,5 × 10 5 cellule staminali etichettati a 26015; g per 5 min a 15 mL provette con 1,5 mL di terreno condrogenica con o senza TGF-β3 (per il campione e controllo, rispettivamente). </li></ol></li><li> Per magneticamente ponteggi seme, inserire ogni scaffold essiccato in una capsula di Petri. Utilizzare polisaccaridi scaffold porosi fatti di pullulano / destrano 21. Per ogni scaffold, diluire 2 x 10 6 cellule staminali marcate in 350 ml di mezzo condrogenica senza TGF-β3 e accuratamente pipetta le cellule sul ponteggio. <ol><li> Incubare per 5 min a 37 ° C per consentire la penetrazione cellulare completo entro l&#39;impalcatura e poi delicatamente aggiungere 3 mL di terreno condrogenica con o senza TGF-β3 (rispettivamente campione o controllo,) alla piastra di Petri. </li><li> Incubare il ponteggio cellularized sul suo dispositivo magnetico a 37 ° C e 5% di CO 2 per 4 giorni per consentire la migrazione cellulare attraverso i pori scaffold e confinamento. </li></ol></li><li> Allo stesso tempo, seminare ponteggi con 2 × 10 6 </sup&gt; cellule staminali etichettati seguendo lo stesso metodo e incubare senza il magnete per ottenere scaffold uniformemente seminato come controlli positivi. </li></ol> 4. differenziazione in condrociti NOTA: Dopo 4 giorni di incubazione, rimuovere i magneti e continuare la maturazione condrogenica sia in una capsula di Petri (condizioni statiche) o in un bioreattore (condizioni dinamiche). campioni di controllo negativi sono maturati in condizioni statiche di media condrogenica senza TGF-β3. <ol><li> In condizioni statiche, mantenere i ponteggi cellularized o aggregati della stessa piastra di Petri. Cambiare il medio condrogenica due volte a settimana per 21 giorni. </li><li> In condizioni dinamiche, preparare il bioreattore. <ol><li> Tagliare tubi in silicone alla lunghezza appropriata secondo il protocollo del produttore. </li><li> Autoclavare tutti materiali: 500 mL camera cultura, tubi, piastre rotanti 2 vie, e gabbie. </li><li> Posizionare le parti in un bioreattore microbio sterilistazione di sicurezza logico. Collegare il tubo per le 2 vie rotatori e alla camera di cultura seguendo le istruzioni del produttore. </li><li> Trasferire accuratamente i ponteggi cellularized in gabbie sterilizzati utilizzando una spatola sterile. Mettere 2 ponteggi per gabbia. Quando le gabbie sono pronte, inserirle nelle aghi del coperchio per impedire loro di muoversi durante un&#39;ulteriore rotazione. Riempire la camera di coltura con terreno condrogenica e chiuderlo con il coperchio contenente le gabbie. </li></ol></li><li> Accendere la pompa peristaltica per riempire il tubo con il mezzo condrogenica e per eliminare le bolle d&#39;aria. </li><li> Posizionare e fissare la camera riempita nel motore del bioreattore e accendere il computer, che controlla le rotazioni sia del braccio e della camera. </li><li> Applicare una velocità di rotazione di 5 rotazioni al minuto (rpm) sia sul braccio e la camera. Regolare la pompa peristaltica ad una portata di 10 rpm per l&#39;alimentazione continua dei ponteggi cellularized. </li></ol> NOTA: Prima di estrazione di RNA, digerire i ponteggi con una soluzione enzimatica. <ol><li> Preparare 1 mL di soluzione enzimatica aggiungendo 100 ml di pullulanasi (40 U / ml) e 50 ml di destranasi (60 mg / ml) a 850 ml di terreno DMEM privo di siero. </li><li> Lavare i ponteggi due volte con DMEM privo di siero, scartare il mezzo, e aggiungere 800 microlitri della soluzione enzimatica per ponteggio. Incubare per 15-30 minuti a 37 ° C sotto leggera agitazione. </li><li> Quando l&#39;impalcatura è completamente sciolto, trasferire la soluzione contenente le cellule ad una provetta da 1,5 mL, centrifugare a 300 xg per 10 min, aspirare accuratamente il mezzo, lavare due volte con sterile 1 × salina tamponata con fosfato (PBS), centrifugare a 300 g per 10 min, e risospendere le cellule in soluzione isolamento dell&#39;RNA. </li><li> Per estrarre l&#39;RNA dagli aggregati, posizionare i sferoidi nella soluzione di isolamento di RNA ecompletamente schiacciare usando un omogeneizzatore prima di eseguire l&#39;estrazione di RNA. </li><li> Isolare l&#39;RNA utilizzando un kit per l&#39;estrazione di RNA totale secondo le istruzioni del produttore. </li><li> Sintetizzare il DNA complementare da 400 ng di RNA totale utilizzando la trascrittasi inversa secondo le istruzioni del produttore, utilizzando 250 ng di primer casuali, 1 ml di miscela di dNTP (10 mM ciascuno), e 40 U / ml RNasi inibitore; il volume finale della reazione è di 20 microlitri. Al termine della reazione, aggiungere 80 ml di acqua distillata per ottenere un volume finale di 100 microlitri. </li><li> Per la reazione a catena della polimerasi (PCR) quantitativa, usare una miscela di PCR contenente un reagente fluorescente per quantificare l&#39;espressione relativa dei geni di interesse, come aggrecan (AGC) e collagene II (Col II), con 10 × diluito cDNA. Normalizzare i livelli di espressione genica con la Ribosomale proteina del gene di riferimento, di grandi dimensioni, P0 (RPLP0). Eseguire i calcoli con il 2 - formula ΔΔCT, Dove ΔΔCT = ΔCT condizione differenziata - significa ΔCT di condizione di controllo, e ogni ΔCT rappresenta la TC del gene di interesse - CT del gene di riferimento (RPLP0). </li><li> Determinare misurazioni statistiche come i valori medi ± l&#39;errore standard della media (SEM). Eseguire l&#39;analisi con n ≥ 2 esperimenti indipendenti. Utilizzare test t di analizzare le differenze statistiche tra pellets centrifugati e fusioni magnetici (* p &lt;0,05). Determinare la significatività con un test di Kruskal-Wallis (ANOVA non parametrico) per analizzare le differenze statistiche tra scaffold differenziati e con lo scaffold controllo (* p &lt;0,05). </li></ol> 6. analisi istologica <ol><li> Lavare i ponteggi cellularized o aggregati con sterile 1 × PBS, fissarli in formalina al 10% per 1 ora a temperatura ambiente, e risciacquare con 1 × PBS. </li><li> Rimuovere la PBS, incorporare i campioni in cuttin ottimalecomposto g temperatura (OCT), e congelarli in un bagno isopentano immersi in azoto liquido. Conservare il campione a -20 ° C. Tagliare i campioni con un criostato avere criosezioni 8 micron per gli aggregati o 12 micron per i ponteggi cellularized. </li><li> Macchiare le criosezioni con 0,5% toluidina soluzione blu per 2 min, risciacquo in acqua di rubinetto, disidratare con etanolo al 100%, chiarire con toluene, e montare i vetrini con un mezzo di montaggio per la microscopia ottica. </li></ol>

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	<li>Naumova, A. V., Modo, M., Moore, A., Murry, C. E., Frank, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+imaging+in+regenerative+medicine.">Clinical imaging in regenerative medicine.</a> Nat Biotechnol. 32 (8), 804-818 (2014).</li><li>Edmundson, M., Thanh, N. T., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+based+stem+cell+tracking+in+regenerative+medicine.">Nanoparticles based stem cell tracking in regenerative medicine.</a> Theranostics. 3 (8), 573-582 (2013).</li><li>Di Corato, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+cellular+MRI:+gadolinium+and+iron+oxide+nanoparticles+for+in-depth+dual-cell+imaging+of+engineered+tissue+constructs.">High-resolution cellular MRI: gadolinium and iron oxide nanoparticles for in-depth dual-cell imaging of engineered tissue constructs.</a> ACS Nano. 7 (9), 7500-7512 (2013).</li><li>Xu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+magnetic+assembly+of+microscale+hydrogels.">Three-dimensional magnetic assembly of microscale hydrogels.</a> Adv Mater. 23 (37), 4254-4260 (2011).</li><li>Kito, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cell+sheets+created+by+a+novel+magnetite+tissue+engineering+method+for+reparative+angiogenesis.">iPS cell sheets created by a novel magnetite tissue engineering method for reparative angiogenesis.</a> Sci Rep. 3, 1418 (2013).</li><li>Mironov, V., Kasyanov, V., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+in+vascular+tissue+engineering:+from+nanoscaffolding+towards+rapid+vessel+biofabrication.">Nanotechnology in vascular tissue engineering: from nanoscaffolding towards rapid vessel biofabrication.</a> Trends Biotechnol. 26 (6), 338-344 (2008).</li><li>Mattix, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janus+magnetic+cellular+spheroids+for+vascular+tissue+engineering.">Janus magnetic cellular spheroids for vascular tissue engineering.</a> Biomaterials. 35 (3), 949-960 (2014).</li><li>Henstock, J., El Haj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+mechanotransduction+in+therapeutic+MSCs:+can+remotely+controlled+magnetic+nanoparticles+regenerate+bones?.">Controlled mechanotransduction in therapeutic MSCs: can remotely controlled magnetic nanoparticles regenerate bones?.</a> Regen Med. 10 (4), 377-380 (2015).</li><li>Fayol, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+magnetic+forces+to+promote+stem+cell+aggregation+during+differentiation,+and+cartilage+tissue+modeling.">Use of magnetic forces to promote stem cell aggregation during differentiation, and cartilage tissue modeling.</a> Adv Mater. 25 (18), 2611-2616 (2013).</li><li>Bartlett, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+implantation+versus+matrix-induced+autologous+chondrocyte+implantation+for+osteochondral+defects+of+the+knee:+a+prospective,+randomised+study.">Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study.</a> J Bone Joint Surg Br. 87 (5), 640-645 (2005).</li><li>Batty, L., Dance, S., Bajaj, S., Cole, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+implantation:+an+overview+of+technique+and+outcomes.">Autologous chondrocyte implantation: an overview of technique and outcomes.</a> ANZ J Surg. 81, 18-25 (2011).</li><li>Song, L., Baksh, D., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-based+cartilage+tissue+engineering:+cells,+scaffold+and+biology.">Mesenchymal stem cell-based cartilage tissue engineering: cells, scaffold and biology.</a> Cytotherapy. 6 (6), 596-601 (2004).</li><li>Boeuf, S., Richter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenesis+of+mesenchymal+stem+cells:+role+of+tissue+source+and+inducing+factors.">Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors.</a> Stem Cell Res Ther. 1 (4), 31 (2010).</li><li>Kock, L., van Donkelaar, C. C., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+functional+articular+cartilage:+the+current+status.">Tissue engineering of functional articular cartilage: the current status.</a> Cell Tissue Res. 347 (3), 613-627 (2012).</li><li>Schon, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+high-throughput+microtissue+fabrication+process+for+3D+assembly+of+tissue+engineered+cartilage+constructs.">Validation of a high-throughput microtissue fabrication process for 3D assembly of tissue engineered cartilage constructs.</a> Cell Tissue Res. , (2012).</li><li>Robert, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+micro-manipulations+to+probe+the+local+physical+properties+of+porous+scaffolds+and+to+confine+stem+cells.">Magnetic micro-manipulations to probe the local physical properties of porous scaffolds and to confine stem cells.</a> Biomaterials. 31 (7), 1586-1595 (2010).</li><li>Luciani, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+chondrogenesis+within+scaffolds,+using+magnetic+stem+cell+confinement+and+bioreactor+maturation.">Successful chondrogenesis within scaffolds, using magnetic stem cell confinement and bioreactor maturation.</a> Acta Biomaterialia. 37, 101-110 (2016).</li><li>Wilhelm, C., Gazeau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+cell+labelling+with+anionic+magnetic+nanoparticles.">Universal cell labelling with anionic magnetic nanoparticles.</a> Biomaterials. 29 (22), 3161-3174 (2008).</li><li>Fayol, D., Luciani, N., Lartigue, L., Gazeau, F., Wilhelm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+magnetic+nanoparticle+aggregation+and+cellular+uptake:+a+precondition+for+efficient+stem-cell+differentiation+and+MRI+tracking.">Managing magnetic nanoparticle aggregation and cellular uptake: a precondition for efficient stem-cell differentiation and MRI tracking.</a> Adv Healthc Mater. 2 (2), 313-325 (2013).</li><li>Wilhelm, C., Gazeau, F., Bacri, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetophoresis+and+ferromagnetic+resonance+of+magnetically+labeled+cells.">Magnetophoresis and ferromagnetic resonance of magnetically labeled cells.</a> Eur Biophys J. 31 (2), 118-125 (2002).</li><li>Autissier, A., Le Visage, C., Pouzet, C., Chaubet, F., Letourneur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+porous+polysaccharide-based+scaffolds+using+a+combined+freeze-drying/cross-linking+process.">Fabrication of porous polysaccharide-based scaffolds using a combined freeze-drying/cross-linking process.</a> Acta Biomater. 6 (9), 3640-3648 (2010).</li><li>Singh, N., Jenkins, G. J. S., Asadi, R., Doak, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+toxicity+of+superparamagnetic+iron+oxide+nanoparticles+(SPION).">Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION).</a> Nano Reviews. 1, (2010).</li><li>Lee, J. H., Hur, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-free+formation+of+a+millimeter-scale+multicellular+spheroid+with+an+internal+cavity+from+magnetically+levitated+3T3+cells+that+ingested+iron+oxide-containing+microspheres.">Scaffold-free formation of a millimeter-scale multicellular spheroid with an internal cavity from magnetically levitated 3T3 cells that ingested iron oxide-containing microspheres.</a> Biotechnol Bioeng. 111 (5), 1038-1047 (2014).</li><li>Mattix, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+magnetic+cellular+spheroids+as+building+blocks+for+tissue+engineering.">Biological magnetic cellular spheroids as building blocks for tissue engineering.</a> Acta Biomaterialia. 10 (2), 623-629 (2014).</li><li>Mazuel, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+Intracellular+Biodegradation+of+Iron+Oxide+Nanoparticles+Evidenced+Magnetically+at+Single-Endosome+and+Tissue+Levels.">Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single-Endosome and Tissue Levels.</a> ACS Nano. 10 (8), 7627-7638 (2016).</li><li>Kim, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+generation+of+spheroids+using+magnetic+nanoparticles+for+three-dimensional+cell+culture.">High-throughput generation of spheroids using magnetic nanoparticles for three-dimensional cell culture.</a> Biomaterials. 34 (34), 8555-8563 (2013).</li><li>Shimizu, K., Ito, A., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+cell-seeding+into+3D+porous+scaffolds+by+use+of+magnetite+nanoparticles.">Enhanced cell-seeding into 3D porous scaffolds by use of magnetite nanoparticles.</a> J Biomed Mater Res B Appl Biomater. 77 (2), 265-272 (2006).</li><li>Sensenig, R., Sapir, Y., MacDonald, C., Cohen, S., Polyak, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle-based+approaches+to+locally+target+therapy+and+enhance+tissue+regeneration+in+vivo.">Magnetic nanoparticle-based approaches to locally target therapy and enhance tissue regeneration in vivo.</a> Nanomedicine (Lond). 7 (9), 1425-1442 (2012).</li><li>Waldman, S. D., Couto, D. C., Grynpas, M. D., Pilliar, R. M., Kandel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-axial+mechanical+stimulation+of+tissue+engineered+cartilage:+review.">Multi-axial mechanical stimulation of tissue engineered cartilage: review.</a> Eur Cell Mater. 13, 66-74 (2007).</li><li>Takahashi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compressive+force+promotes+sox9,+type+II+collagen+and+aggrecan+and+inhibits+IL-1beta+expression+resulting+in+chondrogenesis+in+mouse+embryonic+limb+bud+mesenchymal+cells.">Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells.</a> J Cell Sci. 111 (14), 2067-2076 (1998).</li><li>Campbell, J. J., Lee, D. A., Bader, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compressive+strain+influences+chondrogenic+gene+expression+in+human+mesenchymal+stem+cells.">Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells.</a> Biorheology. 43, 455-470 (2006).</li><li>Vunjak-Novakovic, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+cultivation+conditions+modulate+the+composition+and+mechanical+properties+of+tissue-engineered+cartilage.">Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.</a> J Orthop Res. 17 (1), 130-138 (1999).</li></ol>

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In primo luogo, perché le tecniche qui presentate si basano su l&#39;internalizzazione delle nanoparticelle magnetiche, una questione importante è il risultato delle nanoparticelle, una volta che localizzano all&#39;interno delle cellule. È vero che le nanoparticelle di ferro possono scatenare tossicità potenziale o capacità di differenziazione ridotta seconda delle loro dimensioni, rivestimento, e il tempo di esposizione 19, 22. Tuttavia, numerosi studi ha...

nanoparticelle magnetiche sono già utilizzati in clinica, come agenti di contrasto per la risonanza magnetica (MRI), e le loro applicazioni terapeutiche tenere in espansione. Ad esempio, è stato recentemente dimostrato che le cellule marcate possono essere manipolati in vivo usando un campo magnetico esterno e possono essere orientati e / o mantenuti in un sito definito di impianto 1, 2, 3. In medicina rigenerativa, possono essere utilizzati per ingegnerizzare tessuti organizzati in vitro 4, tra tessuto vascolare 5, 6, 7, 8 osso, cartilagine e 9. La cartilagine articolare è immersa in un ambiente avascolare, le riparazioni dei componenti della matrice extracellulare molto limitati quando si verificano danni. Per questo motivo, research è attualmente focalizzata sulla progettazione di sostituzioni cartilagine ialina che possono essere impiantati nel sito del difetto. Per produrre una sostituzione autologo, alcuni gruppi di ricerca stanno studiando l&#39;utilizzo di condrociti autologhi come fonte cella 10, 11, mentre altri sottolineano la capacità delle cellule staminali mesenchimali (MSC) a differenziarsi in condrociti 12, 13. In studi precedenti ricapitolato qui, abbiamo scelto MSC, come il loro campionamento del midollo osseo è abbastanza semplice e non richiede il sacrificio di condrociti sani, che rischiano di perdere il loro fenotipo 14. Un primo passo essenziale per iniziare la differenziazione condrogenica delle cellule staminali è il loro condensazione. Aggregati cellulari sono comunemente formate utilizzando centrifugazione o cultura Micromass 15; Tuttavia, questi metodi di condensazione Neither presentare il potenziale per creare raggruppamenti cellulari all&#39;interno scaffold spessa né la possibilità di controllare la fusione degli aggregati. In questo documento, si descrive un approccio innovativo alla condensazione cellule staminali utilizzando marcatura magnetica MSC e l&#39;attrazione magnetica. Questa tecnica è stata dimostrata per formare costrutti 3D-free scaffold attraverso la fusione di aggregati tra loro per ottenere un millimetro scala tessuto cartilagineo 9. semina magnetico di scaffold spessi e grandi ha anche permesso la possibilità di aumentare la dimensione della ingegneria tessutale, realizzazione di una forma più facilmente utile per l&#39;impianto, e diversificare il potenziale per applicazioni cliniche di riparazione della cartilagine. Qui, si dettaglio il protocollo per la semina magnetico di MSC nella scaffold porosi costituiti da polisaccaridi naturali, pullulano, e destrano, scaffold precedentemente utilizzato per confinare cellule staminali 16, 17. differenziazione condrogenico era finally eseguita in un bioreattore per garantire continue dei nutrienti e diffusione del gas nel nucleo matrice degli scaffold seminati con un&#39;alta densità di cellule. Oltre a fornire le sostanze nutrienti, fattori di crescita condrogeniche e gas alle cellule, il bioreattore offerto stimolazione meccanica. Nel complesso, la tecnologia magnetica utilizzata per confinare le cellule staminali, in combinazione con la maturazione dinamica in un bioreattore, può notevolmente migliorare la differenziazione condrogenico.

<table><tbody><tr><td>Iron oxide (maghemite) nanoparticules (&amp;gamma;-Fe&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;)</td><td>PHENIX - University Paris 6</td><td>Made and given by C. M&amp;eacute;nager</td><td>Mean diameter of 8.1 nm and negative surface charge</td></tr><tr><td>Polysaccharide Pullulan/Dextran scaffolds</td><td>LIOAD - University Nantes</td><td>Made and given by C. Le Visage</td><td>Prepared from a 75:25 mixture of pullulan/dextran in alkaline conditions (10&amp;nbsp;M NaOH). Porosity: 185-205&amp;nbsp;&amp;micro;m; Thickness: 7&amp;nbsp;mm; Surface area: 1.8&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>TisXell Regeneration System</td><td>QuinXell Technologies</td><td>QX900-002</td><td>Biaxial bioreactor with 500 mL culture chamber&amp;nbsp;</td></tr><tr><td>Cage for scaffolds: Histosette II M492&amp;nbsp;</td><td>VWR</td><td>720-0909</td><td></td></tr><tr><td>Mesenchymal Stem Cell (MSC)</td><td>Lonza</td><td>PT-2501</td><td>Three independant batches have been used</td></tr><tr><td>MSCGM BulletKit medium</td><td>Lonza</td><td>PT-3001</td><td>For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium</td></tr><tr><td>DMEM with Glutamax I</td><td>Life Technologies</td><td>31966-021</td><td>No sodium pyruvate, no HEPES</td></tr><tr><td>RPMI medium 1640, no Glutamine</td><td>Life Technologies</td><td>31870-025</td><td>No sodium pyruvate, no HEPES</td></tr><tr><td>PBS w/o CaCl&lt;sub&gt;2&lt;/sub&gt; w/o MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Life Technologies</td><td>14190-094</td><td></td></tr><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Penicillin (10,000&amp;nbsp;U/mL) / Streptomycin (10,000&amp;nbsp;&amp;micro;g/mL)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>ITS Premix Universal Culture Supplement (20x)</td><td>Corning</td><td>354352</td><td></td></tr><tr><td>Sodium pyruvate solution 100 mM</td><td>Sigma</td><td>S8636</td><td></td></tr><tr><td>L-Ascorbic Acid 2-phosphate</td><td>Sigma</td><td>A8960</td><td>Prepare the concentrated solution (25 mM) in distilled water extemporaneously</td></tr><tr><td>L-Proline</td><td>Sigma</td><td>P5607</td><td>Prepare the 175 mM stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>Prepare the 1 mM stock solution diluted in Ethanol 100% and store at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>TGF-beta 3 protein 10&amp;nbsp;&amp;micro;g</td><td>Interchim</td><td>30R-AT028</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>VWR</td><td>33615.268</td><td>Prepare the 1&amp;nbsp;M stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Pullulanase from &lt;em&gt;Bacillus acidopullulyticus&lt;/em&gt;</td><td>Sigma</td><td>P2986</td><td></td></tr><tr><td>Dextranase from &lt;em&gt;Chaetomium erraticum&lt;/em&gt;</td><td>Sigma</td><td>D0443</td><td></td></tr><tr><td>NucleoSpin RNA Extraction Kit</td><td>Macherey-Nagel</td><td>740955.5</td><td></td></tr><tr><td>SuperScript II Reverse Transcriptase</td><td>Life Technologies</td><td>18064-014</td><td></td></tr><tr><td>Random Primer - Hexamer&amp;nbsp;</td><td>Promega</td><td>C1181</td><td>500 &amp;micro;g/mL: Use diluted 1/2 and put 1 &amp;micro;L per sample</td></tr><tr><td>Recombinant RNAs in ribonuclease inhibitor</td><td>Promega</td><td>N2511</td><td>40 U/&amp;micro;L: put 1 &amp;micro;L per sample</td></tr><tr><td>PCR nucleotide dNTP mix (10&amp;nbsp;mM each)</td><td>Roche</td><td>10842321</td><td></td></tr><tr><td>SyBr Green PCR Master Mix</td><td>Life Technologies</td><td>4368708</td><td></td></tr><tr><td>Step One Plus Real-Time PCR System</td><td>Life Technologies</td><td>4381792</td><td></td></tr><tr><td>Formalin solution 10% neutral buffered</td><td>Sigma</td><td>HT5012</td><td></td></tr><tr><td>OCT solution</td><td>VWR</td><td>361603E</td><td></td></tr><tr><td>Isopentane</td><td>Sigma</td><td>M32631</td><td></td></tr><tr><td>Toluidine blue O</td><td>VWR</td><td>1.15930.0025</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td>20821.310</td><td></td></tr><tr><td>Toluene</td><td>VWR</td><td>1.08323.1000</td><td></td></tr><tr><td>Mounting medium Pertex</td><td>Histolab</td><td>840</td><td></td></tr><tr><td>RPLP0 Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TGCATCAGTAC&lt;br/&gt; CCCATTCTATCAT-3&#039;;&lt;br/&gt; 5&#039;-AAGGTGTAATC&lt;br/&gt; CGTCTCCACAGA-3&#039;</td></tr><tr><td>Aggrecan Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TCTACCGCTGCGAGGTGAT-3&#039;; 3&#039;-TGTAATGGAACACGATGCCTTT-5&#039;</td></tr><tr><td>Collagen II Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-ACTGGATTGACCCCAACCAA-3&#039;; 3&#039;-TCCATGTTGCAGAAAACCTTCA-5&#039;</td></tr></tbody></table>

3d magnetic stem cell aggregation and bioreactor maturation for cartilage regeneration

Ingegneria cartilagine rimane una sfida a causa delle difficoltà nel creare un impianto funzionale in vitro simile al tessuto nativo. Un approccio recentemente esplorato per lo sviluppo delle sostituzioni autologhe comporta la differenziazione delle cellule staminali in condrociti. Per avviare questo condrogenesi, un grado di compattazione delle cellule staminali è richiesta; quindi, abbiamo dimostrato la fattibilità di cellule magneticamente condensazione, sia all&#39;interno scaffold spessi e privo di scaffold, utilizzando miniaturizzati sorgenti di campo magnetico come attrattori cellulari. Questo approccio magnetico è stato utilizzato anche per guidare fusione aggregato e costruire privo di impalcatura, organizzato, tridimensionale (3D) i tessuti diversi millimetri di dimensione. Oltre ad avere una dimensione maggiore, il tessuto formato da fusione magnetica guidato presentato un significativo aumento dell&#39;espressione di collagene II, ed una tendenza simile è stato osservato per l&#39;espressione aggrecan. Come la cartilagine nativa è stato sottoposto a forze tcappello influenzato la sua struttura 3D, la maturazione dinamica è stata effettuata anche. Un bioreattore che fornisce stimoli meccanici stato usato per cultura scaffold magneticamente seminate su un periodo di 21 giorni. Bioreattore maturazione in gran parte migliorato chondrogenesis nelle impalcature cellularized; la matrice extracellulare ottenuto in queste condizioni era ricca di collagene II e aggrecano. Questo lavoro illustra il potenziale innovativo di condensazione magnetica delle cellule staminali etichettati e maturazione dinamica in un bioreattore per una migliore differenziazione condrogenica, sia-impalcatura libero e all&#39;interno polisaccaridi ponteggi.

In primo luogo, gli aggregati possono essere formati individualmente tramite microorganismi magneti depositando 2,5 x 10 5 cellule staminali marcate (Figura 2A). Questi singoli aggregati (~ 0,8 mm di dimensione) possono essere fusi in strutture più grandi grazie sequenziale, fusione indotta magneticamente. Per esempio, il giorno 8 di maturazione condrogenica, aggregati sono stati posti in contatto in coppia per formare doppietti; quartine sono state assemblat...

Watch this Scientific Journal Video about 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration at JoVE.com

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

Condrogenesi da cellule staminali richiede la messa a punto delle condizioni di coltura. Qui vi presentiamo un approccio magnetico per condensare le cellule, un passo essenziale per iniziare condrogenesi. Inoltre, mostriamo che la maturazione dinamico in un bioreattore applica stimolazione meccanica ai costrutti cellulari e aumenta la produzione di matrice extracellulare cartilaginea.

3D Stem magnetica cellulare Aggregazione e bioreattore maturazione per la cartilagine Regeneration

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

3d-magnetic-stem-cell-aggregation-bioreactor-maturation-for-cartilage

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.7K Views.  UMR 7057 CNRS and University Paris Diderot.  Condrogenesi da cellule staminali richiede la messa a punto delle condizioni di coltura. Qui vi presentiamo un approccio magnetico per condensare le cellule, un passo essenziale per iniziare condrogenesi. Inoltre, mostriamo che la maturazione dinamico in un bioreattore applica stimolazione meccanica ai costrutti cellulari e aumenta la produzione di matrice extracellulare cartilaginea. 

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

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Progettazione di un biassiale Carico meccanico bioreattore per l&#39;ingegneria tissutale

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Ricerca

Bioingegneria

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Formazione di pellet condensato da cellule staminali pluripotenti indotte dal sangue

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Biologia dello sviluppo

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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.

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 Hydrogel Ponteggi per articolare condrociti, Cultura e Cartilagine Generation

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

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

L&#39;impianto di Ferumoxides etichetta cellule staminali mesenchimali in difetti della cartilagine

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Biologia

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.

Multimateriali di cartilagine e pelle tessuto analoghi utilizzando un romanzo passivo unità tecnica di miscelazione per Bioink Precellularization

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

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.

Coltura di cellule di mammifero in scaffold tridimensionale Peptide

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

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

Applicazione di un sistema di bioreattore tridimensionale di stimolazione meccanica uniassiale per indurre la differenziazione tenogena delle cellule staminali derivate da Tendon

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

Medicina

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.

Generazione di sferoidi da vari condrociti utilizzando condizioni di bassa adesività per gravità e mini-bioreattori fatti in casa

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

3D Stem magnetica cellulare Aggregazione e bioreattore maturazione per la cartilagine Regeneration

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Progettazione di un biassiale Carico meccanico bioreattore per l'ingegneria tissutale