printing-thermoresponsive-reverse-molds-for-creation-patterned-two

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Cartilage Engineering & Regeneration

Il destino delle cellule &egrave; regolato da segnali dell&#39;ambiente extracellulare. Interazione specifico recettori della cellula con proteine, glicani, fattori solubili, come pure le cellule vicine pu&ograve; dirigere le cellule verso la proliferazione, differenziazione, apoptosis o migrazione. In questa recensione, sono descritti gli approcci per costruire strutture cellulari di ingegneria gli aspetti dell&#39;ambiente extracellulare. Questi metodi includono modifiche non specifici per controllare la bagnabilit&agrave; e la rigidit&agrave; delle superfici utilizzando monostrati auto-assemblati (SAMs) e multistrati polielettrolita (PEM), nonch&eacute; metodi dove viene controllato l&#39;attivazione temporale e distribuzione spaziale dei ligandi di adesione. Basandosi su queste tecniche, costruzione di fogli di cella bidimensionale utilizzando polimeri sensibili di temperatura o dissoluzione elettrochimica &egrave; descritto insieme ad applicazioni correnti di questi innesti in ambito clinico. Infine, metodi per celle modello in tre dimensioni come pure per quanto riguarda strumentalizzano l&#39;ambiente 3D con motivi biologici portarci un passo pi&ugrave; vicino ad essere in grado di ingegnere di organi e tessuti multicellulari.

Ingegneria dell&#39;ambiente extracellulare: strategie per la costruzione di strutture cellulari 2D e 3D.

Cell fate is regulated by extracellular environmental signals. Receptor specific interaction of the cell with proteins, glycans, soluble factors as well as neighboring cells can steer cells towards proliferation, differentiation, apoptosis or migration. In this review, approaches to build cellular structures by engineering aspects of the extracellular environment are described. These methods include non-specific modifications to control the wettability and stiffness of surfaces using self-assembled monolayers (SAMs) and polyelectrolyte multilayers (PEMs) as well as methods where the temporal activation and spatial distribution of adhesion ligands is controlled. Building on these techniques, construction of two-dimensional cell sheets using temperature sensitive polymers or electrochemical dissolution is described together with current applications of these grafts in the clinical arena. Finally, methods to pattern cells in three-dimensions as well as to functionalize the 3D environment with biologic motifs take us one step closer to being able to engineer multicellular tissues and organs.

Engineering the extracellular environment: Strategies for building 2D and 3D cellular structures.

Sort de la cellule est r&eacute;gul&eacute;e par des signaux extracellulaires environnementales. L&#39;interaction sp&eacute;cifique du r&eacute;cepteur de la cellule avec les prot&eacute;ines, glycanes, facteurs solubles ainsi que les cellules voisines peut orienter vers la prolif&eacute;ration, la diff&eacute;renciation, l&#39;apoptose ou la migration des cellules. Dans cette revue, les approches pour construire des structures cellulaires par les aspects techniques de l&#39;environnement extracellulaire sont d&eacute;crites. Ces m&eacute;thodes incluent des modifications non sp&eacute;cifique pour contr&ocirc;ler la mouillabilit&eacute; et la rigidit&eacute; des surfaces &agrave; l&#39;aide des monocouches auto-assembl&eacute;es (SAMs) et multicouches de poly&eacute;lectrolytes (SEMP) ainsi que les m&eacute;thodes o&ugrave; l&#39;activation temporelle et la distribution spatiale des ligands d&#39;adh&eacute;rence est contr&ocirc;l&eacute;e. S&#39;appuyant sur ces techniques, construction de feuilles cellulaire &agrave; deux dimensions &agrave; l&#39;aide de polym&egrave;res sensibles de temp&eacute;rature ou de la dissolution &eacute;lectrochimique est d&eacute;crit ainsi que les applications actuelles de ces greffes dans le domaine clinique. Enfin, m&eacute;thodes aux cellules du mod&egrave;le en trois dimensions ainsi quant &agrave; FONCTIONNALISER l&#39;environnement 3D avec des motifs biologiques nous prennent un peu plus qu&#39;il est capable &agrave; l&#39;ing&eacute;nieur d&#39;organes et tissus multicellulaires.

Ing&eacute;nierie de l&#39;environnement extracellulaire : strat&eacute;gies pour le renforcement des structures cellulaires 2D et 3D.

This article describes a dynamic platform in which the biointerfacial properties of micro-patterned domains can be switched electrochemically through the spatio-temporally controlled dissolution and adsorption of polyelectrolyte coatings. Insulating SU-8 micro-patterns created on a transparent indium tin oxide electrode by photolithography allowed for the local control over the electrochemical dissolution of polyelectrolyte mono- and multilayers, with polyelectrolytes shielded from the electrochemical treatment by the underlying photoresist stencil. The platform allowed for the creation of micro-patterned cell co-cultures through the electrochemical removal of a non-fouling polyelectrolyte coating and the localized adsorption of a cell adhesive one after attachment of the first cell population. In addition, the use of weak adhesive polyelectrolyte coatings on the photoresist domains allowed for the detachment of a contiguous heterotypic cell sheet upon electrochemical trigger. Cells grown on the ITO domains peeled off upon electrochemical dissolution of the sacrificial polyelectrolyte substrate, whereas adjacent cell areas on the insulated weakly adhesive substrate easily detached through the contractile force generated by neighboring cells. This electrochemical strategy for the micro-patterning and detachment of heterotypic cell sheets combines simplicity, precision and versatility, and presents great prospects for the creation of cellular constructs which mimic the cellular complexity of native tissues.

Electrochemically switchable platform for the micro-patterning and release of heterotypic cell sheets.

Cet article d&eacute;crit une plate-forme dynamique dans lequel les propri&eacute;t&eacute;s biointerfacial des domaines micro &agrave; motifs peuvent &ecirc;tre commut&eacute;es &eacute;lectrochimiquement par le biais de la dissolution spatio-temporel contr&ocirc;l&eacute;e et l&#39;adsorption des rev&ecirc;tements de poly&eacute;lectrolyte. Isolant SU-8 micro-mod&egrave;les cr&eacute;&eacute;s sur une &eacute;lectrode d&#39;oxyde d&#39;&eacute;tain indium transparent par photolithographie autoris&eacute;e pour le contr&ocirc;le local de la dissolution &eacute;lectrochimique de poly&eacute;lectrolyte mono et multicouches, avec poly&eacute;lectrolytes prot&eacute;g&eacute;s contre le traitement &eacute;lectrochimique par le gabarit de photor&eacute;sine sous-jacent. La plateforme a permis la cr&eacute;ation des co-cultures aux motifs micro cellulaire par le biais de l&#39;enl&egrave;vement &eacute;lectrochimique d&#39;un rev&ecirc;tement antisalissure non de poly&eacute;lectrolyte et l&#39;adsorption localis&eacute;e d&#39;un adh&eacute;sif de cellule un apr&egrave;s fixation de la premi&egrave;re population de cellules. En outre, l&#39;utilisation de rev&ecirc;tements de poly&eacute;lectrolyte adh&eacute;sif faible sur les domaines de la r&eacute;sine photosensible autoris&eacute;s pour le d&eacute;tachement d&#39;une feuille de cellule h&eacute;t&eacute;rotypiques contigus apr&egrave;s d&eacute;clenchement &eacute;lectrochimique. Les cellules cultiv&eacute;es sur les domaines de l&#39;ITO &eacute;caill&eacute; &agrave; la dissolution &eacute;lectrochimique du substrat poly&eacute;lectrolyte sacrificiel, tandis que les zones de la cellule adjacente sur le substrat adh&eacute;sif faiblement isol&eacute; d&eacute;tach&eacute; facilement par le biais de la force contractile g&eacute;n&eacute;r&eacute;e par les cellules voisines. Cette strat&eacute;gie &eacute;lectrochimique pour le micro-structuration et le d&eacute;tachement des feuilles de cellule h&eacute;t&eacute;rotypiques allie simplicit&eacute;, pr&eacute;cision et polyvalence et pr&eacute;sente de grandes perspectives pour la cr&eacute;ation de constructions cellulaires qui imitent la complexit&eacute; cellulaire des tissus natifs.

Plate-forme &eacute;lectrochimiquement commutable pour les micro-structuration et la sortie de cellule h&eacute;t&eacute;rotypiques feuilles.

Questo articolo descrive una piattaforma dinamica in cui le propriet&agrave; biointerfacial di micro-fantasia domini possono essere commutate elettrochimicamente attraverso la dissoluzione controllata spazio-temporale e l&#39;adsorbimento dei rivestimenti di polielettrolita. Isolante 8 SU micro-modelli creato su un elettrodo di ossido di stagno trasparente indio di fotolitografia ammessi per il controllo locale sulla dissoluzione elettrochimica di polielettrolita mono e multistrato, con polielettroliti schermati dal trattamento elettrochimico di stencil photoresist sottostante. La piattaforma ha permesso la creazione di micro-fantasia cell co-cultures attraverso la rimozione elettrochimica di un rivestimento di polielettrolita non fouling e l&#39;adsorbimento localizzata di un adesivo cella uno dopo attaccamento della popolazione cellulare prima. Inoltre, l&#39;uso di rivestimenti di polielettrolita adesivo debole sui domini photoresist consentito per il distacco di un foglio di celle contigue heterotypic su trigger di elettrochimica. Cellule coltivate sui domini ITO sfil&ograve; sulla dissoluzione elettrochimica del substrato polielettrolita sacrificale, mentre aree di celle adiacenti sul substrato adesivo debolmente isolato staccato facilmente attraverso la forza contrattile generata da celle vicine. Questa strategia elettrochimica per la micro-patterning e distacco dei fogli heterotypic cella combina semplicit&agrave;, precisione e versatilit&agrave; e presenta grandi prospettive per la creazione di costrutti cellulari che mimano la complessit&agrave; cellulare dei tessuti nativi.

Piattaforma elettrochimicamente commutabile per la micro-patterning e il rilascio dei fogli heterotypic cell.

Terapie del peptide sono di crescente interesse a causa della loro specificit&agrave; biologica. Abbiamo utilizzato una tecnica semplice per studiare l&#39;efficacia di induzione di peptidi in condrociti aderenti da permeabilizing transitoriamente la membrana con impulsi elettrici (elettroporazione in situ). Meccano-growth factor (MGF) &egrave; stato selezionato come un peptide modello. Marcato con FITC MGF &egrave; stato aggiunto a colture di condrociti primari aderenti coltivati su vetrini rivestito ITO. Le cellule sono stati sottoposti ad impulsi 3-9 di 175-275 V e valutate in citometria a flusso. In condizioni ottimali, un&#39;efficienza di elettroporazione di quasi il 50% potrebbe essere raggiunto. Questa tecnica pu&ograve; essere utilizzata per studiare i domini funzionali di peptidi intracellulare, inibizione del peptide della trasduzione del segnale e intracrine-mediata effetti dei peptidi in cellule aderenti.

Elettroporazione efficace dei peptidi in cellule aderenti: indagine del ruolo del fattore di crescita mechano nella coltura di condrociti.

Peptide therapeutics are of increasing interest due to their biological specificity. We used a simple technique to study the efficacy of inducing peptides into adherent chondrocytes by transiently permeabilizing the membrane with electric pulses (in situ electroporation). Mechano-growth factor (MGF) was selected as a model peptide. FITC-labeled MGF was added to cultures of adherent primary chondrocytes grown on ITO coated glass slides. Cells were subjected to 3-9 pulses of 175-275 V and evaluated by flow cytometry. Under optimal conditions, an electroporation efficiency of close to 50% could be achieved. This technique can be used to study the functional domains of intracellular peptides, peptide inhibition of signal transduction and intracrine-mediated effects of peptides in adherent cells.

Efficient electroporation of peptides into adherent cells: investigation of the role of mechano-growth factor in chondrocyte culture.

Th&eacute;rapeutique de peptide sont de plus en plus en raison de leur sp&eacute;cificit&eacute; biologique. Nous avons utilis&eacute; une technique simple pour &eacute;tudier l&#39;efficacit&eacute; d&#39;induction des peptides en chondrocytes adh&eacute;rentes de perm&eacute;abiliser transitoirement la membrane avec des impulsions &eacute;lectriques (&eacute;lectroporation in situe). M&eacute;cano-growth factor (MGF) a &eacute;t&eacute; choisi comme un peptide de mod&egrave;le. Marqu&eacute; FITC MGF a &eacute;t&eacute; ajout&eacute; aux cultures des chondrocytes de primaires adh&eacute;rentes cultiv&eacute;es sur lames de verre enduit de ITO. Cellules ont &eacute;t&eacute; soumis &agrave; des impulsions de 3-9 de 175-275 V et &eacute;valu&eacute;es par cytom&eacute;trie en flux. Dans des conditions optimales, une efficacit&eacute; d&#39;&eacute;lectroporation de pr&egrave;s de 50 % pourrait &ecirc;tre atteint. Cette technique permet d&#39;&eacute;tudier les domaines fonctionnels des peptides intracellulaires, l&#39;inhibition de peptide de transduction de signal et intracrine-effets des peptides dans les cellules adh&eacute;rentes.

&Eacute;lectroporation efficace des peptides dans les cellules adh&eacute;rentes : &eacute;tude du r&ocirc;le du m&eacute;cano-facteur de croissance en culture de chondrocytes.

The layer-by-layer (LbL) technique has been widely used to produce nanofilms for biomedical applications. Naturally occurring polymers such as ECM macromolecules are attractive candidates for LbL film preparation. In this study, we assessed the build-up of type I collagen (Col1)/chondroitin sulfate (CS) or Col1/Heparin (HN) on polydimethylsiloxane (PDMS) substrates. The build-up was assessed by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Integrin-mediated cell adhesion was assessed by studying the cytoskeletal organization of mammalian primary cells (chondrocytes) seeded on different end layers and number of layers. Data generated from the QCM-D observations showed a consistent build-up of films with more adsorption in the case of Col1/HN. Col1/CS films were stable in media, whereas Col1/HN films were not. AFM analysis showed that the layers were fibrillar in structure for both systems and between 20 and 30 nm thick. The films promoted cell adhesion when compared with tissue culture plastic in serum-free media with cycloheximide. Crosslinking of the films resulted in constrained cell spreading and a ruffled morphology. Finally, beta1 integrin blocking antibodies prevented cell spreading, suggesting that cell adhesion and spreading were mediated mainly by interaction with the collagen fibrils. The ability to construct stable ECM-based films on PDMS has particular relevance in mechanobiology, microfluidics, and other biomedical applications.

Layer-by-layer films made from extracellular matrix macromolecules on silicone substrates.

La technique de couche par couche (LbL) a &eacute;t&eacute; employ&eacute; couramment pour produire nanofilms pour applications biom&eacute;dicales. Polym&egrave;res naturels tels que les macromol&eacute;cules de l&#39;ECM sont candidats attrayants pour la pr&eacute;paration du film LbL. Dans cette &eacute;tude, nous avons &eacute;valu&eacute; l&#39;accumulation de collag&egrave;ne (Col1) de type I / chondro&iuml;tine sulfate (CS) ou Col1/h&eacute;parine (HN) sur des substrats de polydim&eacute;thylsiloxane (PDMS). L&#39;accumulation a &eacute;t&eacute; &eacute;valu&eacute;e par microbalance &agrave; cristal de quartz avec dissipation (QCM-D) et la microscopie &agrave; force atomique (AFM). Adh&eacute;sion int&eacute;grine m&eacute;diation cellulaire a &eacute;t&eacute; &eacute;valu&eacute;e par l&#39;&eacute;tude de l&#39;organisation du cytosquelette des cellules primaires de mammif&egrave;res (chondrocytes) ensemenc&eacute;s sur des calques diff&eacute;rents fin et nombre de couches. Donn&eacute;es g&eacute;n&eacute;r&eacute;es &agrave; partir des observations de QCM-D ont montr&eacute; une accumulation uniforme de films avec plus d&#39;adsorption dans le cas de Col1/HN. Col1/CS films &eacute;taient stables dans les m&eacute;dias, alors que les films de Col1/HN n&#39;&eacute;taient pas. Analyse de l&#39;AFM a d&eacute;montr&eacute; que les couches fibrillaires de structure pour les deux syst&egrave;mes et entre 20 et 30 nm d&#39;&eacute;paisseur. Les films promu adh&eacute;sion cellulaire en comparaison avec du plastique de culture de tissus dans des milieux sans s&eacute;rum avec le cycloheximide. R&eacute;ticulation des films a abouti &agrave; la diffusion de cellule contrainte et une morphologie &eacute;bouriff&eacute;e. Enfin, beta1 int&eacute;grine blocage anticorps emp&ecirc;ch&eacute; cellule &eacute;pandeuses, sugg&eacute;rant que l&#39;adh&eacute;sion cellulaire et propagation ont &eacute;t&eacute; v&eacute;hicul&eacute;es principalement par l&#39;interaction avec les fibrilles de collag&egrave;ne. La capacit&eacute; &agrave; construire des films &agrave; base de ECM stables sur PDMS a une importance particuli&egrave;re dans la m&eacute;canobiologie, microfluidique et autres applications biom&eacute;dicales.

Par-couche t&eacute;l&eacute;films de macromol&eacute;cules de la matrice extracellulaire sur des substrats de silicone.

La tecnica di strato per strato (LbL) &egrave; stato ampiamente usata per produrre nanofilms per applicazioni biomediche. Polimeri naturali come macromolecole ECM sono candidati attraenti per la preparazione di film LbL. In questo studio abbiamo valutato l&#39;accumulo di collagene (Col1) di tipo I / Condroitin solfato (CS) o Col1/eparina (HN) su substrati di polidimetilsilossano (PDMS). L&#39;accumulo &egrave; stato valutato dalla microbilancia al quarzo cristallo con dissipazione (QCM-D) e microscopia a forza atomica (AFM). Adesione integrina-mediata delle cellule &egrave; stata valutata studiando l&#39;organizzazione del citoscheletro delle cellule di mammiferi primaria (condrociti) seminato su fine diversi strati e numero di strati. Dati generati dalle osservazioni QCM-D ha mostrate un consistente accumulo di pellicole con adsorbimento pi&ugrave; nel caso Col1/HN. Col1/CS film erano stabili nei media, considerando che non erano film Col1/HN. AFM analisi ha mostrato che i livelli erano fibrillare in struttura per entrambi i sistemi e tra 20 e 30 nm di spessore. Il film promosso adesione cellulare quando confrontato con la coltura del tessuto plastica media serum-free con cicloeximide. Reticolazione del film ha portato in cella vincolata diffusione e una morfologia arruffata. Infine, beta1 integrina bloccando gli anticorpi impedito cella diffusione, suggerendo che adesione cellulare e diffusione erano mediato principalmente dall&#39;interazione con le fibrille di collagene. L&#39;abilit&agrave; di costruire stabili film ECM basato su PDMS ha particolare rilevanza in mechanobiology, microfluidica e altre applicazioni biomediche.

Strato dopo strato di film realizzati da macromolecole della matrice extracellulare su substrati in silicone.

The enormous progress of nanotechnology during the last decade has made it possible to fabricate a great variety of nanostructures. On the nanoscale, metals exhibit special electrical and optical properties, which can be utilized for novel applications. In particular, plasmonic sensors including both the established technique of surface plasmon resonance and more recent nanoplasmonic sensors, have recently attracted much attention. However, some of the simplest and most successful sensors, such as the glucose biosensor, are based on electrical readout. In this review we describe the implementation of electrochemistry with plasmonic nanostructures for combined electrical and optical signal transduction. We highlight results from different types of metallic nanostructures such as nanoparticles, nanowires, nanoholes or simply films of nanoscale thickness. We briefly give an overview of their optical properties and discuss implementation of electrochemical methods. In particular, we review studies on how electrochemical potentials influence the plasmon resonances in different nanostructures, as this type of fundamental understanding is necessary for successful combination of the methods. Although several combined platforms exist, many are not yet in use as sensors partly because of the complicated effects from electrochemical potentials on plasmon resonances. Yet, there are clearly promising aspects of these sensor combinations and we conclude this review by discussing the advantages of synchronized electrical and optical readout, illustrating the versatility of these technologies.

Electrochemical plasmonic sensors.

Les &eacute;normes progr&egrave;s de la nanotechnologie au cours de la derni&egrave;re d&eacute;cennie a permis de fabriquer une grande vari&eacute;t&eacute; de nanostructures. &Agrave; l&#39;&eacute;chelle nanom&eacute;trique, m&eacute;taux pr&eacute;sentent des propri&eacute;t&eacute;s &eacute;lectriques et optiques sp&eacute;ciales, qui peuvent &ecirc;tre utilis&eacute;es pour nouvelles applications. En particulier, les capteurs plasmoniques, y compris les deux &eacute;tablie la technique de r&eacute;sonance plasmonique de surface et de la plus r&eacute;centes nanoplasmonic capteurs, ont r&eacute;cemment attir&eacute; beaucoup d&#39;attention. Toutefois, certains des capteurs plus simples et la plus r&eacute;ussies, comme le biocapteur de glucose, reposent sur l&#39;affichage &eacute;lectrique. Dans cette revue, nous d&eacute;crivons la mise en &oelig;uvre de l&#39;&eacute;lectrochimie avec nanostructures plasmoniques pour la transduction des signaux &eacute;lectriques et optiques combin&eacute;s. Nous mettons en &eacute;vidence les r&eacute;sultats des diff&eacute;rents types de nanostructures m&eacute;talliques tels que les nanoparticules, nanofils, nanoholes ou tout simplement les films d&#39;&eacute;paisseur nanom&eacute;trique. Bri&egrave;vement, nous donnent un aper&ccedil;u de leurs propri&eacute;t&eacute;s optiques et discuter la mise en &oelig;uvre de m&eacute;thodes &eacute;lectrochimiques. En particulier, nous examinons des &eacute;tudes sur l&#39;influence de potentiels &eacute;lectrochimiques comment la r&eacute;sonance plasmon dans les nanostructures diff&eacute;rents, car ce type de compr&eacute;hension fondamentale est n&eacute;cessaire pour une combinaison r&eacute;ussie des m&eacute;thodes. Bien que plusieurs plateformes combin&eacute;s existent, beaucoup sont pas encore utilis&eacute;s comme capteurs en partie en raison des effets complexes de potentiels &eacute;lectrochimiques sur plasmon r&eacute;sonances. Pourtant, il y a clairement promettant des aspects de ces combinaisons de capteur et nous terminons cette revue en discutant les avantages de lecture synchronis&eacute;e d&#39;&eacute;lectrique et optique illustrant la polyvalence de ces technologies.

Capteurs &eacute;lectrochimiques plasmoniques.

Gli enormi progressi della nanotecnologia durante l&#39;ultimo decennio ha reso possibile fabbricare una grande variet&agrave; di nanostrutture. Su scala nanometrica, metalli esibiscono propriet&agrave; elettriche e ottiche speciali, che possono essere utilizzati per nuove applicazioni. In particolare, plasmoniche sensori inclusi sia la tecnica stabilita di risonanza plasmonica di superficie e pi&ugrave; recenti nanoplasmonic sensori, recentemente hanno attirato molta attenzione. Tuttavia, alcuni dei sensori pi&ugrave; semplici e di maggior successo, come ad esempio il biosensore glucosio, sono basate sulla lettura elettrica. In questa rassegna descriveremo l&#39;implementazione di elettrochimica con nanostrutture plasmoniche per la trasduzione del segnale elettrico ed ottico combinato. Si evidenziano risultati da diversi tipi di nanostrutture metalliche come ad esempio le nanoparticelle, nanofili, nanoholes o semplicemente film di spessore su scala nanometrica. Brevemente diamo una panoramica delle loro propriet&agrave; ottiche e discutere l&#39;implementazione dei metodi elettrochimici. In particolare, passiamo in rassegna gli studi sull&#39;influenza di potenziali elettrochimici come la risonanza plasmonica in diverse nanostrutture, come questo tipo di comprensione fondamentale &egrave; necessario per la riuscita combinazione di metodi. Sebbene esistano diverse piattaforme combinate, molti non sono ancora in uso come sensori in parte a causa degli effetti complicati da potenziali elettrochimici su plasmon risonanze. Eppure, ci sono chiaramente promettendo aspetti di queste combinazioni di sensore e Concludiamo questa recensione di discutere i vantaggi della lettura sincronizzata elettrico e ottico, illustrando la versatilit&agrave; di queste tecnologie.

Sensori elettrochimici plasmoniche.

In the field of human mesenchymal stromal cell (MSC) research, quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the method of choice to study changes in gene expression patterns upon differentiation, application of stimuli, or of factors such as inhibitors or siRNAs. To reliably detect small changes, the use of a reference gene (RG) that is stably expressed under all conditions is essential. The large number of different RGs used in the field and the lack of validation of their suitability make the comparison between studies impossible. Therefore, this work aims to establish one single RG for mesodermal differentiation studies that use MSCs. Seven commonly used RGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal protein L13a [RPL13a], beta-actin [ACTB], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide [YWHAZ], eukaryotic translational elongation factor 1 alpha [EF1Î±], Î²2-microglobulin [B2M], and 18S ribosomal RNA [18S]) were investigated concerning their mRNA expression stability during expansion of bone marrow-derived MSCs up to four passages as well as during their adipo-, chondro-, and osteogenenic differentiation on days 9, 16, and 22 after induction. RPL13a was validated for qPCR studies of MSCs (bone marrow- and placenta-derived) and, additionally, for primary human bone cells (HBCs) and the osteosarcoma cell line MG-63. GAPDH and ACTB, the two most frequently used RGs, showed the highest expression variance. The superior performance of RPL13a should make it the RG of choice for all MSC studies addressing mesodermal differentiation.

Ribosomal protein l13a as a reference gene for human bone marrow-derived mesenchymal stromal cells during expansion, adipo-, chondro-, and osteogenesis.

Dans le domaine de la recherche de cellules stromales m&eacute;senchymateuses humaines (MSC), quantitative reverse transcription-PCR en temps r&eacute;el (qPCR) est la m&eacute;thode de choix pour &eacute;tudier les changements dans les profils d&#39;expression g&eacute;nique sur la diff&eacute;renciation, application de stimuli ou de facteurs tels que des inhibiteurs ou des siARN. Pour d&eacute;tecter des petits changements de fa&ccedil;on fiable, l&#39;utilisation d&#39;un g&egrave;ne de r&eacute;f&eacute;rence (RG) qui s&#39;exprime de fa&ccedil;on stable dans toutes les conditions est essentielle. Le grand nombre de RGs diff&eacute;rents utilis&eacute;s dans le domaine et l&#39;absence de validation de leurs qualit&eacute;s font la comparaison entre les &eacute;tudes impossibles. Par cons&eacute;quent, ces travaux visent &agrave; &eacute;tablir un RG unique pour les &eacute;tudes de diff&eacute;renciation m&eacute;sodermiques qui utilisent MSCs. sept couramment utilis&eacute; RGs (glyc&eacute;rald&eacute;hyde-3-phosphate d&eacute;shydrog&eacute;nase [GAPDH], prot&eacute;ine ribosomique L13a [RPL13a], beta-actine [ACTB], prot&eacute;ine de tyrosine 3-monooxyg&eacute;nase/tryptophane 5-monooxyg&eacute;nase activation, zeta-polypeptide [YWHAZ], facteur 1 d&#39;&eacute;longation translationnelle eucaryotes alpha [EF1&alpha;], &beta;2-microglobuline [B2M] et 18 s ribosomal RNA [18]) ont &eacute;t&eacute; &eacute;tudi&eacute;s concernant leur stabilit&eacute; d&#39;expression ARNm durant l&#39;expansion de la moelle osseuse MSCs jusqu&#39;&agrave; quatre passages, ainsi que pendant leur adipo -, chondro-et la diff&eacute;renciation d&#39;osteogenenic 9, 16 et 22 jours apr&egrave;s l&#39;induction. RPL13a a &eacute;t&eacute; valid&eacute;e pour les &eacute;tudes de qPCR de MSCs (moelle osseuse et placenta-d&eacute;riv&eacute;s) et, en outre, pour les cellules des os humains primaires (pays les plus touch&eacute;s) et la lign&eacute;e de cellules d&#39;ost&eacute;osarcome MG-63. GAPDH et ACTB, les deux utilis&eacute;s plus fr&eacute;quemment RGs, a montr&eacute; la plus forte variance d&#39;expression. La performance sup&eacute;rieure de RPL13a devrait faire le RG de choix pour toutes les &eacute;tudes de Master traitant de diff&eacute;renciation m&eacute;sodermiques.

Prot&eacute;ine ribosomique l13a comme un g&egrave;ne de r&eacute;f&eacute;rence pour la moelle osseuse m&eacute;senchymateuses stromales cellules humaines pendant expansion, adipo - chondro- et ost&eacute;ogen&egrave;se.

Nel campo della ricerca sulle cellule stromali mesenchimali umane (MSC), quantitative Real-Time reverse transcription-polymerase chain reaction (qPCR) &egrave; il metodo di scelta per studiare i cambiamenti nei modelli di espressione genica su differenziazione, applicazione di stimoli, o fattori inibitori o siRNA. Per rilevare in modo affidabile i piccoli cambiamenti, &egrave; indispensabile l&#39;uso di un gene di riferimento (RG) che &egrave; stabilmente espressa in tutte le condizioni. Il gran numero di differenti RGs utilizzato in campo e la mancanza di convalida della loro idoneit&agrave; fare il confronto tra studi impossibili. Quindi, questo lavoro mira a stabilire una RG unico per studi di differenziazione mesodermica che utilizzano MSCs. sette comunemente usato RGs (gliceraldeide-3-fosfato deidrogenasi [GAPDH], proteina ribosomiale L13a [RPL13a], [ACTB] beta-actina, proteina di attivazione tirosina 3-monoossigenasi/triptofano 5-monoossigenasi, zeta-polipeptide [YWHAZ], alfa di fattore 1 di allungamento eucariotico traslazionale [EF1&alpha;], &beta;2-microglobulina [B2M] e 18S RNA ribosomiale (rRNA) [18]) sono stati investigati riguardo la loro stabilit&agrave; di espressione del mRNA durante l&#39;espansione di MSCs derivate dal midollo osseo fino a quattro passaggi, cos&igrave; come durante la loro adipo -, Condro-e osteogenenic differenziazione nei giorni 9, 16 e 22 dopo l&#39;induzione. RPL13a &egrave; stato convalidato per qPCR studi di MSCs (midollo e placenta-derivati) e, inoltre, per le cellule di osso umano primario (carcinomi) e la linea di cellule di osteosarcoma MG-63. GAPDH e ACTB, le due pi&ugrave; frequentemente utilizzati RGs, ha mostrato la varianza massima espressione. Le prestazioni superiori di RPL13a dovrebbero rendere il RG di scelta per tutti gli studi MSC indirizzamento mesodermica differenziazione.

Proteina ribosomiale l13a come un gene di riferimento per umane derivate da midollo osseo mesenchymal cellule stromali durante l&#39;espansione, adipo-, Condro- e osteogenesi.

Chondrocyte hypertrophy is one of the key physiological processes involved in the longitudinal growth of long bones, yet the regulation of hypertrophy is also becoming increasingly relevant for clinical application of mesenchymal stem cells (MSCs) and screening for drugs to treat hypertrophic osteoarthritis. The extraordinary cell volume increase during hypertrophy is accompanied by an up-regulation of collagen X, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF), all which are targets of the runt-related transcription factor 2 (Runx2). Many pathways, including parathyroid hormone-related protein (PTHrP)/Indian Hedgehog, Wingless/Int (Wnt)/Î²-catenin, and transforming growth factor beta (TGF-Î²)/Sma and Mad Related Family (Smad) pathways, can regulate hypertrophy, but factors as diverse as hypoxia, co-culture, epigenetics and biomaterial composition can also potently affect Runx2 expression. Control of hypertrophic differentiation can be exploited both for cartilage repair, where a stable phenotype is desired, but also in bone regeneration, where hypertrophic cartilage could act as a template for endochondral bone formation. We hope this review will motivate the design of novel engineered microenvironments for skeletal regeneration applications.

Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells.

Hypertrophie du chondrocyte est l&#39;un des principaux processus physiologiques impliqu&eacute;s dans la croissance longitudinale des os longs, mais le r&egrave;glement de l&#39;hypertrophie est aussi devient de plus en plus pertinent pour l&#39;application clinique des cellules souches m&eacute;senchymateuses (CSM) et d&eacute;pistage de m&eacute;dicaments pour traiter l&#39;arthrose hypertrophique. L&#39;augmentation de volume des cellules extraordinaire au cours de l&#39;hypertrophie est accompagn&eacute;e d&#39;une r&eacute;gulation du collag&egrave;ne X, m&eacute;talloprot&eacute;inases matricielles (MMP) et vascular endothelial growth factor (VEGF), tout ce qui sont les cibles du facteur de transcription li&eacute;s avorton 2 (Runx2). Plusieurs voies, y compris parathyroid hormone-related protein (PTHrP) / Indian Hedgehog, voie Wingless/Int (Wnt) / &beta;-cat&eacute;nine et transformer la croissance facteur b&ecirc;ta (TGF-&beta;) / Sma et Mad voies famille apparent&eacute;e (Smad), permet de r&eacute;gler l&#39;hypertrophie, mais des facteurs aussi divers que la composition de l&#39;hypoxie, co-culture, &eacute;pig&eacute;n&eacute;tique et biomat&eacute;riaux peuvent affecter aussi puissamment Runx2 expression. Contr&ocirc;le de la diff&eacute;renciation hypertrophique peut &ecirc;tre exploit&eacute;e tant pour la r&eacute;paration du cartilage, o&ugrave; un ph&eacute;notype stable est souhait&eacute;, mais aussi dans la r&eacute;g&eacute;n&eacute;ration osseuse, o&ugrave; le cartilage hypertrophique pourrait agir comme un mod&egrave;le pour l&#39;ossification endochondrale. Nous esp&eacute;rons que cet examen va motiver la conception de nouveaux microenvironnements machin&eacute;s pour des applications de r&eacute;g&eacute;n&eacute;ration squelettique.

M&eacute;canismes mol&eacute;culaires et biophysiques hypertrophique diff&eacute;renciation dans les chondrocytes et de cellules souches m&eacute;senchymateuses de r&eacute;gulation.

Condrociti ipertrofia &egrave; uno dei processi fisiologici chiavi coinvolti nella crescita longitudinale delle ossa lunghe, ma il regolamento dell&#39;ipertrofia &egrave; anche diventando sempre pi&ugrave; rilevanti per l&#39;applicazione clinica delle cellule staminali mesenchimali (MSCs) e lo screening per farmaci trattare l&#39;osteoartrosi ipertrofica. L&#39;aumento del volume cellulare straordinario durante l&#39;ipertrofia &egrave; accompagnato da una up-regolazione di collagene X, metalloproteinasi della matrice (MMP) e vascular endothelial growth factor (VEGF), che sono gli obiettivi del fattore di trascrizione correlati a runt 2 (Runx2). Numerosi sentieri, tra cui l&#39;ormone paratiroideo-related protein (PTHrP) / Indian Hedgehog, Gabrile/Int (Wnt) / &beta;-catenina e trasformare la crescita fattore beta (TGF-&beta;) / percorsi di famiglia correlati (Smad) Sma e Mad, pu&ograve; regolare l&#39;ipertrofia, ma fattori diversi come l&#39;ipossia, co-culture, epigenetica e biomateriale composizione possono interessare anche potentemente Runx2 espressione. Controllo della differenziazione ipertrofica pu&ograve; essere sfruttata sia per la riparazione della cartilagine, dove un fenotipo stabile &egrave; desiderato, ma anche nella rigenerazione ossea, dove la cartilagine ipertrofica potrebbe agire come un modello per la formazione di osso endocondrale. Speriamo che questa recensione motiver&agrave; il design del romanzo microambienti ingegnerizzati per applicazioni di rigenerazione ossea.

Meccanismi molecolari e biofisici che regolano la differenziazione ipertrofica nei condrociti e di cellule staminali mesenchimali.

Neurite formation and synaptic patterning are fundamental to the development of a functional nervous system. Flavonoids are natural molecules known for having beneficial effects on brain health through diverse molecular pathways. Cytoskeletal changes occurring during neuritogenesis and synapse formation often involve Rho GTPases. Here we hypothesized that the flavonoid isoquercitrin promotes neuronal differentiation through Rho signalling.

The flavonoid isoquercitrin promotes neurite elongation by reducing RhoA activity.

Neurite formation et structuration synaptique sont fondamentales pour le d&eacute;veloppement d&#39;un syst&egrave;me nerveux fonctionnel. Les flavono&iuml;des sont des mol&eacute;cules naturelles, connus pour avoir des effets b&eacute;n&eacute;fiques sur la sant&eacute; du cerveau par le biais de divers m&eacute;canismes mol&eacute;culaires. Les modifications du cytosquelette survenant au cours de la formation neuritogenesis et synapse souvent comportent des GTPases Rho. Ici nous avons &eacute;mis l&#39;hypoth&egrave;se que le flavono&iuml;des Isoquercitrine favorise la diff&eacute;renciation neuronale par signalisation de Rho.

Le flavono&iuml;des Isoquercitrine favorise les neurites &eacute;longation en r&eacute;duisant l&#39;activit&eacute; de RhoA.

Formazione dei neuriti e modellatura sinaptica sono fondamentali per lo sviluppo di un sistema nervoso funzionale. I flavonoidi sono molecole naturali noti per avere effetti benefici sulla salute del cervello attraverso diverse vie molecolari. Citoscheletriche cambiamenti che si verificano durante la formazione del neuritogenesis e sinapsi spesso coinvolgono Rho GTPases. Qui abbiamo ipotizzato che i flavonoidi isoquercitrina promuove la differenziazione neuronale attraverso la segnalazione di Rho.

Article	Year
Engineering the extracellular environment: Strategies for building 2D and 3D cellular structures. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 20842659	2010
Electrochemically switchable platform for the micro-patterning and release of heterotypic cell sheets. Biomedical microdevices\| PubMed ID: 21057978	2011
Efficient electroporation of peptides into adherent cells: investigation of the role of mechano-growth factor in chondrocyte culture. Biotechnology letters\| PubMed ID: 21207114	2011
Layer-by-layer films made from extracellular matrix macromolecules on silicone substrates. Biomacromolecules\| PubMed ID: 21319812	2011
Electrochemical plasmonic sensors. Analytical and bioanalytical chemistry\| PubMed ID: 21947010	2012
Ribosomal protein l13a as a reference gene for human bone marrow-derived mesenchymal stromal cells during expansion, adipo-, chondro-, and osteogenesis. Tissue engineering. Part C, Methods\| PubMed ID: 22533734	2012
Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells. European cells & materials\| PubMed ID: 22828990	2012
The flavonoid isoquercitrin promotes neurite elongation by reducing RhoA activity. PloS one\| PubMed ID: 23209630	2012

Marcy Zenobi-Wong

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