5% GTA/0.1 M phosphate buffered saline (PBS) at room temperature for 2 h. After washing twice with 0.1 M PBS, the cells were postfixed with 1% osmium tetroxide at temperature for 1 h. The cells were then washed twice with PBS, dehydrated through serial gradients of Aurora Kinase inhibitor ethanol (10 min per each gradient), and finally dried out by the critical point dryer Bal-Tec CPD-030 (Bal-Tec AG, Balzers, Liechtenstein).

The cells along with the substrates were sputtered with gold at a current of 15 mA for 3 min by the ion sputter EMITECH K575X. SEM imaging was conducted at voltages ranging from 5 to 10 kV. Staining on actin and nuclei and fluorescence confocal microscopy HAECs were cultured on the functionalized pSi substrates for 48 h. After cell culture experiments, culture media were removed and cells were washed two times with PBS at 37°C. The cells were fixed with a 4% (w/v) solution of paraformaldehyde in PBS for 30 min at room temperature. After washing two times more with PBS, the substrates were immersed in 0.2% Triton-X 100 in PBS for 10 min at room temperature to permeabilize the cell membrane. After rinsing with PBS two times, the actin filaments and nuclei were stained in the dark at room temperature. Actin-stain 670 phalloidin (tebu-bio, Le Perray-en-Yvelines, France)

was used to stain the actin filaments (200 nM, 30 min), while NucGreen Dead 488 (Life Technologies, Carlsbad, CA, USA) was used to stain the nuclei (two drops/mL, 10 min). Each sample was washed three times with PBS, and after mounting on microscope slides selleck kinase inhibitor using anti-fade mounting media, the samples were incubated overnight in the dark at room temperature. Stained cells were kept at 4°C in the dark until microscope observations. The fluorescence images were acquired using a Nikon Eclipse TE2000-E inverted

microscope (Nikon Instruments, Amsterdam, Netherlands), equipped with a C1 laser confocal system (EZ-C1 software, Nikon). Argon 488- and 633-nm lasers were used as excitation sources for NucGreen and phalloidin, respectively. Results and discussion The porous silicon (pSi) samples were produced by electrochemical etching of p-type silicon wafers in HF-based electrolytes [22]. Two types of samples were generated by varying the etching conditions in order to study the cellular response on surfaces with different pore geometry. PSi substrates obtained from Methamphetamine silicon wafers with a resistivity of 0.002 to 0.004 Ω cm by applying a constant current density of 60 mA/cm2 had an average pore diameter of 30 to 50 nm. The pSi produced from silicon wafers with 10 to 20 Ω cm resistivity, by applying a current density of 4 mA/cm2, had an average pore diameter of 1 to 1.5 μm. The topography of theses substrates was analyzed using scanning electron microscopy. Figure  1a,b shows representative images of the top view of macro- and nanoporous substrates, which were surface-modified by oxidation and silanization with APTES to promote cell adhesion.