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IL-1β secretion from peripheral blood mononuclear cells (1.10 6 cells/ml) in experiments that were carried out using (A) unfiltered TCM or (B) 0.2-μm filtered TCM. Influence of experimental conditions on IL-1β responses to apatitic nanoparticles.
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(H) Infrared analysis of hydroxyapatite standard (Sigma 0–200-nm nanopowder) and (I) infrared analysis of the in situ-formed calcium phosphate particles in tissue culture medium with spectral features attributed as follows: (a) lattice vibrations, (b) phosphate vibration, (c) carbonate adsorption bands at 1465–1410 cm -1, (d) amine adsorption bands from serum proteins at 1600–16,700 cm -1, and (e) probable OH broadening from residual water with the main OH band at 3400 cm -1.ĭLS: Dynamic light scattering NTA: Nanoparticle tracking analysis SLS: Static light scattering T3: 3 h T8: 8 h T24: 24 h. Physicochemical characterization of in situ-formed calcium phosphate nanoparticles.įollowing synthesis, calcium phosphate particles were analyzed (A–C) for particle size and structure by transmission electron microscopy, for size distribution in tissue culture medium by (D) DLS, (E) NTA and (F) static light scattering, and (G) for elemental composition by energy dispersive x-ray spectroscopy within the transmission electron microscopy (C and Cu signals are generated by the support film and grid). Original submitted 13 September 2013 Revised submitted 6 March 2014įigure 1.
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However, the experimental setting must be very carefully considered as it may promote false-positive outcomes. Conclusion: In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. Results: Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. Material & methods: The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Aim: To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles.