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Additional file 1: Figure S1. Vascular grafts fabricated with cellularized tubular HA/collagen nanofibrous scaffold facilitates vascular reconstruction. H&E staining the cross-section of a retrieved tubular HA/collagen nanofibrous grafts 6 weeks after transplant at 2.5 $$\times $$ �� (upper panel) and 40 $$\times $$ �� (lower panel) compared with rabbit carotid artery (b, d, g). Scale bars, 100 ��m. Table S1. The size of the obtained electrospun tubular HA/collagen nanofibrous scaffolds.

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions., This work was supported by grants to D.N., T.L., N.C., and J.J.U. (Spanish Ministry of Economy and Competitiveness FIS-PI14/00280), M.L. and J.B. (National Science Center NCN DEC-2011/01/M/ST3/00711), H.O. (Deutsche Forschungsgemeinschaft, Koselleck 68/1 and Centre of Excellence, Cells in Motion (CIM), University of Münster, Germany), S.B. and F.L. (ANR-10-EQPX-04-01) and A.D., E.T.H., R.G. (CSD2010-00024, ERC-AdG-340177). A.P. thanks the Dept. of Physics of the University of Milano for financial support under the project “Piano di Sviluppo dell’Ateneo per la Ricerca 2014”, actions: “A. Upgrade of instrumentation”, and “B. Support to young researchers”. We acknowledge the support of the International AFMBioMed Conference Association.

Room-Temperature Ionic Liquids (ILs) have attracted considerable interest in recent years. This interest is motivated by the physico-chemical properties of these systems, tunable modifying the chemical structure of ions. Generally, ILs show chemical and thermal stability, i.e. they do not easily decompose or react. Furthermore, these compounds remain liquid over an extended range of temperatures, in which they show also a remarkably low volatility. The low vapor pressure of ILs, promote them as good solvents for the growing field of the ?Green Chemistry?, in substitution of the volatile organic compounds. The fact that ILs are composed solely by ions, and can have a quite wide electrochemical window, make them very interesting as electrolytes. For these purposes, this PhD thesis is devoted to the investigation of ILs in contact with solid interfaces, primary targets of interaction. To deepen the analysis of electric properties at the solid interface, thin layers of ILs deposited on conductive substrates were investigated by means of AFM. The ?Green? character of these compounds was investigated studying their interaction with biomembrane models and external membranes of living cells, by means of AFM and electrochemical methods. Because of their ionic nature, ILs can be used as electrolytes in several devices aimed at conversion and storage of energy, such as electrochemical supercapacitors, Graetzel solar cells and batteries. In these devices a key role is played by the interface between the surface of the electrodes and the electrolyte; in particular, structural-morphological and electrical properties of the first few nanometers of IL interacting with the solid electrode surface are expected to have the strongest impact on device performance. AFM morphological analysis of small quantity of [C 4 MIM] [NTf 2 ] IL, deposited on various insulating surfaces revealed a population of nanodroplets and new structures. Remarkably, the solid surfaces induce the organization of the ionic liquid into regular, lamellar solid-like nanostructures presenting a high degree of vertical structural order and high mechanical resistance to normal compressive stresses. Nanomechanical investigation reveals that the structures resist to normal compressive loads up to 1.5 MPa; beyond that limit, indentation, in discrete steps, occurs. Furthermore, lamellar [C 4 MIM] [NTf 2 ] islands are not affected when scanned by a biased AFM tip under the influence of an electric field as intense as 10 8 V/m, while the liquid nano-and micro-droplets are easily swept away. These results confirm the solid-like character of the ordered lamellar nanostructures observed when thin films of [C 4 MIM] [NTf 2 ] are deposited on solid surfaces, and suggest that these films may possess an insulating, dielectric behavior, at odd with the case of the bulk ionic liquids. Nanoscale impedance measurements (capacitance vs. distance) and electrostatic force spectroscopy (electric force vs. distance) between a conductive AFM tip and the IL structures confirmed that values of the dielectric constant (? r = 3-5) are significantly smaller than those measured in the bulk liquid (? r = 9-15). These results support the picture of solid-like ordered domains where the ion mobility is significantly inhibited with respect to the bulk liquid phase. These findings also highlight the potentialities of scanning probe techniques for the quantitative investigation of the interfacial electrical properties of thin ionic liquid films, suggesting that ILs at electrified solid surfaces may possess unexpected electrical and structural properties, thus influencing the behavior of photo-electrochemical devices. The ?green? character of ionic liquids (ILs) is dependent on their negligible vapor pressure but in contrast to their environmental behavior their intrinsic toxicity is not at present completely understood. Accordingly, although ILs will not evaporate which alleviates air pollution problems, a potential hazard of Ils to living organisms via aqueous media cannot be ruled out. A rigorous investigation on the interaction of ILs with biomaterials is required to provide information about their intrinsic toxicity. In order to test the biocompatible character of ILs, as a first objective, the interaction of various ILs with biological membrane (biomembrane) models was studied using electrochemical methods. A series of imidazolium based ILs were investigated whose interactions highlighted the role of anion and lateral side chain of cation during the interaction with dioleoyl phosphatidylcholine (DOPC) monolayer. It was shown that the hydrophobic and lipophilic character of the IL cations is a primary factor responsible for this interaction. The modifications of the Hg supported monolayer caused by ILs were simultaneously monitored electrochemically in a well controlled manner using rapid cyclic voltammetry (RCV), alternating current voltammetry (ACV), and electrochemical impedance spectroscopy (EIS). Hg supported monolayers provide an accurate analysis of the behavior of ILs at the interface of a biomembrane leading to a comprehensive understanding of the interaction mechanisms involved. At the same time, these experiments show that the Hg-phospholipid model is an effective toxicity sensing technique as shown by the correlation between literature in vivo toxicity data and the data from this study. Cell membrane is the main target of ILs interaction, depending on the lipophilicity of hydrophobic lateral chain of cation. In order to test the biocompatible character of ILs, the interaction of various imidazolium-based ILs with supported DOPC phospholipid bilayers (as models of the cell membrane) and living MDA-MB-231 cells (@37 ?C) was investigated. Atomic Force Microscopy (AFM) was used to carry on a combined topographic and mechanical analysis of supported DOPC bilayers as well as of living cells. During the analysis of DOPC bilayers we have observed modifications in breakthrough force and membrane elasticity related to the ingress of lateral chains of cations in the bilayer, demonstrating agreement with electrochemical results. The parallel nanomechanical analysis performed on living cells interacting with ILs at various concentrations showed modifications of elasticity (effective Young?s modulus) and morphology of cells after exposure to ILs dispersed in their culture medium. The measurements confirmed the primary action of ILs on membrane and actin cytoskeleton, highlighting a subtoxic/toxic effect dependent on ILs concentration and chemical nature of cation. Our results may be helpful for filling existing gaps of knowledge about ionic liquids toxicity and their impact on living organisms. From these evidences, interaction of ILs with micro-organisms and single cells is an important step to assess the environmental sustainability of this novel and promising class of solvents and to attribute a ?green? label to it. Studying the interaction of ionic liquids, it has been recognized that the interface is a vital component. When the bulk symmetry of IL is broken by surfaces, the electrical properties are greatly affected, leading from a ion conductor to an insulator behavior. Also the interaction with biological entity is driven, in first instance, by surface interaction. Biomembrane models and cell membranes are affected by ILs that accumulate/penetrate the surface interface, leading to structural reorganization/damage of external membrane.