1,720,995 research outputs found
Mechanosensing of substrate stiffness regulates focal adhesions dynamics in cell
No full textCell mechanical recognition of extracellular matrix determines the cell activities and functions. Focal adhesions are part of the cell mechanosensing machinery and, operating at the very dynamic interface between cell and extracellular matrix, can operate this recognition and trigger conformational, functional and behavioral modification of the cell. To investigate how the dynamic of assembly and disassembly of focal adhesion are influenced by the substrate mechanics we developed a novel procedure. The analysis consists of the over time tracking of focal adhesion structures in a stable cell line of NIH/3T3 expressing fluorescent pmKate2-paxillin. From collected signals and by their autocorrelation we evaluated the average lifetime and assembly rate of focal adhesion as function of substrate stiffness. Further, by signals cross-correlation we obtained information about the mechanical nature of cytoskeleton and its network. This quantitative approach to focal adhesion dynamics characterization was presented in this study as an investigation tool for cell mechanobiology
Crosstalk between focal adhesions and material mechanical properties governs cell mechanics and functions
No full textMechanical properties of materials strongly influence cell fate and functions. Focal adhesions are involved in the extremely important processes of mechanosensing and mechanotransduction. To address the relationship between the mechanical properties of cell substrates, focal adhesion/cytoskeleton assembly and cell functions, we investigated the behavior of NIH/3T3 cells over a wide range of stiffness (3-1000 kPa) using two of the most common synthetic polymers for cell cultures: polyacrylamide and polydimethylsiloxane. An overlapping stiffness region was created between them to compare focal adhesion characteristics and cell functions, taking into account their different time-dependent behavior. Indeed, from a rheological point of view, polyacrylamide behaves like a strong gel (elastically), whereas polydimethylsiloxane like a viscoelastic solid. First, focal adhesion characteristics and dynamics were addressed in terms of material stiffness, then cell spreading area, migration rate and cell mechanical properties were correlated with focal adhesion size and assembly. Focal adhesion size was found to increase in the whole range of stiffness and to be in agreement in the overlapping rigidity region for the investigated materials. Cell mechanics directly correlated with focal adhesion lengths, whereas migration rate followed an inverse correlation. Cell spreading correlated with the substrate stiffness on polyacrylamide hydrogel, while no specific trend was found on polydimethylsiloxane. Substrate mechanics can be considered as a key physical cue that regulates focal adhesion assembly, which in turn governs important cellular properties and functions
ECM mechanoregulation in malignant pleural mesothelioma
No full textMalignant pleural mesothelioma is a relatively rare, but devastating tumor, because of the difficulties in providing early diagnosis and effective treatments with conventional chemo- and radiotherapies. Patients usually present pleural effusions that can be used for diagnostic purposes by cytological analysis. This effusion cytology may take weeks or months to establish and has a limited sensitivity (30%–60%). Then, it is becoming increasingly urgent to develop alternative investigative methods to support the diagnosis of mesothelioma at an early stage when this cancer can be treated successfully. To this purpose, mechanobiology provides novel perspectives into the study of tumor onset and progression and new diagnostic tools for the mechanical characterization of tumor tissues. Here, we report a mechanical and biophysical characterization of malignant pleural mesothelioma cells as additional support to the diagnosis of pleural effusions. In particular, we examined a normal mesothelial cell line (Met5A) and two epithelioid mesothelioma cell lines (REN and MPP89), investigating how malignant transformation can influence cellular function like proliferation, cell migration, and cell spreading area with respect to the normal ones. These alterations also correlated with variations in cytoskeletal mechanical properties that, in turn, were measured on substrates mimicking the stiffness of patho-physiological ECM
New insights into the mechanisms of the interactions between doxorubicin and the ion-exchange hydrogel DC BeadTM for use in transarterial chemoembolization (TACE).
No full textIon-exchange microspheres (IEMs) are widely employed in controlled drug delivery of ionic drugs due to their high loading capacity and the possibility to obtain the controlled release of the loaded drug(s) at a specific site. Among IEMs, DC Bead(TM) are embolic microdevices (100-300 μm diameter) designed for transarterial chemoembolization (TACE) and composed of cross-linked poly(vinyl alcohol) (PVA) hydrogel, bearing anionic sulfonate moieties on the cross-links, and able to bind cationic drugs such as doxorubicin hydrochloride (Dox). Even if DC Bead(TM) were studied for their release and bulk characteristics, a thorough characterization of these devices is still lacking. In particular, the aim of this work was the determination of bound and free water, Dox distribution within the microdevices and drug-DC Bead(TM) interactions, in terms of transport features within the device. Compared with previous results, different Dox radial distributions in DC Bead(TM) were found, and related to bead microsctructure and ion exchange mechanism. Artifacts due to the self-quenching of Dox at high concentration were prevented and the diffusion coefficients of drug-polymer (Dox-ionic sites) evaluated in different sections of the microspheres. Furthermore, DSC results indicated that in the hydrogel either free (bulk) or bound (non-freezable) water could be found, and that no freezing-bound water was present
Stimuli-responsive chitosan/poly (N-isopropylacrylamide) semi-interpenetrating polymer networks: effect of pH and temperature on their rheological and swelling properties
No full textThe aim of this work was to synthesize semi-interpenetrating polymer networks (semi-IPNs) by free radical polymerization of N-isopropylacrylamide [poly (NIPAAm)], in the presence of chitosan (CHI), and to study the effect of pH and temperature changes on their rheological and swelling properties. The semi-IPNs are thermally stable up to about 400 °C and the presence of CHI increases the thermal degradation rate compared to bare poly (NIPAAm). The prepared systems presents a well-defined porosity and proved to be non-toxic, in vitro, on human embryonic skin fibroblast, thus offering appropriate support for cell proliferation. The semi-IPNs present, at physiological pH, swelling degrees well below those of the pure poly (NIPAAm). Differently, at acidic pH, the CHI macromolecules are protonated and become much more permeable to the diffusion of water giving a swelling degree that approaches that of bare poly (NIPAAm). The viscoelastic moduli of the semi-IPNs increase as a function of pH while the LCST remain unchanged. Moreover, the semi-IPNs viscoelastic moduli increase with the increase of CHI content and, in particular, the difference between the elastic modulus before and after the sol/gel transition is higher for the semi-IPN than for bare poly (NIPAAm) just at about physiological conditions. © 2016, Springer Science+Business Media New York
Effetti di nanoparticelle di polistirene sull'embriogenesi di Xenopus laevis
No full textL'impiego di nanoparticelle (NP) utili per diverse applicazioni (cosmetica, abbigliamento, alimentazione e drug delivery) riscuote un vivo interesse. In medicina offrono la possibilità unica di superare la barriera cellulare per dirigere molecole verso targets specifici come le droghe utilizzate in chemioterapia. Per promuovere il giusto sviluppo di tali tecnologie è essenziale chiarire le potenziali conseguenze per la salute umana associate all???esposizione alle NP. La principale preoccupazione riguarda le loro piccole dimensioni e la possibilità che possano essere internalizzate inadeguatamente, in rapporto con le loro caratteristiche fisico-chimiche e con la natura delle cellule target (Shawna et al., 2011; Gorth et al., 2011; Pompa et al., 2011). Le NP di polistirene da noi utilizzate sono considerate materiale ???biologicamente inerte. L'impiego del sistema modello Xenopus laevis, permette di saggiare l???eventuale tossicità delle NP in vivo, valutandone gli effetti sullo sviluppo embrionale. A questo scopo sono state somministrate con due diversi protocolli: la microiniezione in uno dei blastomeri di embrioni allo stadio 2 cellule e il contatto che consiste nell'allevamento di embrioni in 1/10 Ringer contenente NP. Per entrambi i tipi di esperimenti sono state analizzate la morfologia, la percentuale di mortalità e l'espressione di alcuni marcatori embrionali quali: bra (mesoderma presuntivo), myod1 (mesoderma parassiale) e sox9 (creste neurali). Gli embrioni sia microiniettati che contatto presentano malformazioni di testa, intestino e coda, edemi variamente diffusi e mostrano, inoltre, uno sviluppo più lento. Gli embrioni microiniettati presentano un alto tasso di mortalità mentre quelli a contatto hanno un tasso di mortalità simile ai w.t. La microscopia confocale ha evidenziato la presenza di NP nell'intestino, nella faringe e nello strato esterno della retina, non nel cervello. I marcatori usati presentano tutti modificazioni dell'espressione. Questi dati suggeriscono un effetto tossico/teratogenico ma non letale delle NP per gli embrioni trattati alle condizioni precedentemente descritte
Xenopus laevis come modello per lo studio degli effetti tossici da nanoparticelle
No full textNuovi nanomateriali (NM) vengono continuamente prodotti e prontamente utilizzati per varie applicazioni, tra cui: cosmesi, cura personale, elettronica e sistemi di drug delivery. In particolare, in medicina, le nanoparticelle (NP) offrono un'opportunità unica per superare le barriere fisiologiche e fornire agenti bioattivi alle cellule. Attualmente, la comunità scientifica dedica molti sforzi allo sviluppo sostenibile di questa tecnologia. Tuttavia, l'uso di NM potrebbe essere pericoloso per cui si presta molta attenzione ai loro potenziali effetti tossici sugli organismi viventi. Evidenze sperimentali mostrano che le NP possono essere associate a un aumento del rischio di alcune malattie per l???uomo a causa della loro tossicità e il rischio aumenta con la riduzione della loro dimensione. Questa nuova forma di inquinamento presenta un grave problema per la comunità data la mancanza di procedure standard volte a certificare la loro attività nelle complesse interazioni che si verificano tra NM e sistemi viventi. È ben noto che gli ambienti acquatici sono certamente a rischio di esposizione a questi inquinanti, in quanto, rappresentano un sito di concentrazione per la maggior parte dei contaminanti ambientali. Il nostro lavoro sfrutta la possibilità di utilizzare Xenopus laevis come modello affidabile e robusto per la valutazione della nanotossicità in organismi acquatici utilizzando nanoparticelle di polistirene (PSNPs), di Fe3O4 (nanoFe) e di SiO2 (nanoSi). Abbiamo utilizzato due procedure di somministrazione: microiniezione allo stadio di 2 cellule e diluizione nel mezzo di coltura. Abbiamo rilevato malformazioni della testa, dell???intestino e della coda in tutti gli embrioni trattati; edema nella zona ventrale anteriore e una lunghezza del corpo inferiore rispetto ai wt. Solo gli embrioni microiniettati mostrano un alto tasso di mortalità. La microscopia confocale ci ha permesso di localizzare le PSNPs nel citoplasma, nel nucleo e nella periferia cellulare. Mentre la microscopia elettronica ha mostrato quadri di sofferenza dell???intestino sia in trattati con nanoSi che con nanoFe. Queste ultime si localizzano a livello dei villi intestinali. Lo studio dell'espressione di geni coinvolti nelle prime fasi dello sviluppo embrionale ha mostrato una distribuzione anomala del marcatore precoce del mesoderma bra, del marcatore del mesoderma parassiale myod1, e di sox9, marcatore delle creste neurali. I nostri dati suggeriscono che la tossicità delle nanoparticelle dipende dalla loro concentrazione nel mezzo e dalla capacità di penetrare in profondità nel tessuto. Inoltre, dimostrano che Xenopus laevis potrebbe essere un potenziale modello per valutare la tossicità delle nanoparticelle a livello quantitativo e qualitativo
Polystyrene nanoparticles affect Xenopus laevis development
No full textExposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay-Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the "corona'' effect caused by the interaction of PSNPs with cytoplasm components. The three end-points of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos
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