The electronic structure of as deposited and annealed ns TiO2 was characterized in a UHV CC-5013 ap paratus Leybold LHS 10 12 equipped with a hemispherical electron analyzer and conventional X ray source. The high resolution spectra were acquired in constant pass energy mode Epass 30 eV with an overall energy resolution of 1. 0 eV. All spectra are referenced to the Fermi level and the binding energy scale is calibrated via the Au 4f5 2 core level line of a clean polycrystalline Au sample. No charging effects on the samples under investigation were observed during all the measurements. The line shapes were fitted with mixed singlets obtained by a linear combination of a Gaussian and a Lorentzian profiles sited on a Shirley background.

Cell culture and analysis Cell culture Rat PC12 cells were used as a model to test nanostructured surface effect on cell differentiation because of their fac ulty to assume neuronal phenotype responding to some stimuli, as. The hu man Inhibitors,Modulators,Libraries neuroblastoma SH SY5Y cell line, which responds Inhibitors,Modulators,Libraries to retinoic acid, chronic NGF or BDNF, has been also used in some experiments. After annealing the glass cover slips coated with ns TiO2 or flat TiO2 were sterilized by expo sure to UV light for 30 min. Sterilized glass pre coated with Poly L Lysine 0. 01% solutions were used as positive controls. PC12 were maintained in RPMI 1640 Medium supplemented with 10% horse serum, 5% fetal bovine serum, 2 mM l glutamine, 100 units mL penicillin, 100 ug mL streptomycin, 1 mM pyruvic acid and 10 mM Hepes in 5% CO2, 98% air humidified incubator at 37 C.

Cells were detached from culture dishes using a solution 1 mM EDTA in HBSS, centrifuged at 1000 Inhibitors,Modulators,Libraries x g for 5 min, and resuspended in culture medium. Subcultures or culture medium exchanges were routinely established every 2nd to 3rd day into Inhibitors,Modulators,Libraries Petri dishes. During the experiment the PC12 were suspended in low serum medium added with 50 ng mL NGF, 2 mM S methylisothiourea. 10 uM U0126 and control sol vent where specified, and seeded at a cell density of 5 20 104 cm2 for nitration, proliferation, neurite Inhibitors,Modulators,Libraries and NOS inhibi tor analysis. Following seeding, cells were maintained in 5% CO2, 98% air humidified incubator at 37 C, and the medium was exchanged every 24 and 48 h after Phos phate Buffered Saline wash. For nitration analysis, cells were seeded on rectangular glass slides and cul tured into 4 well rectangular dishes.

For all other analyses, cells were seeded on round cover glass and cultured into 24 well test plates. SH SY5Y cells were maintained in RPM1 supplemented with 10% FCS, 1% pen strep and CHIR99021 molecular weight 1% L glu either on glass coverslips or nanostructured sub strates, in the absence of growth factors. To label neurites, immunocytochemical staining for the protein Synaptosomal associated protein 25 was carried out, using described methods.