After static or dynamic immersion, the samples were removed from the solutions, washed with distilled water and selleck chemicals then dried in air, under sterile hood. For every characterization, the pristine TCP and TCP-T plates were used as controls. Surface characterization after biomimetic immersion study The morphology of TCP and TCP-T after biomimetic immersion study was examined by scanning electron microscopy (SEM) in a JEOL JSM 6460LV microscope to investigate the surface transformations. The analysis was done once and the most representative pictures of each samples were selected. The analysis of the surface chemistry was performed in the same time using an EDX system coupled to the scanning electron microscope. XPS X-ray photoelectron spectroscopy (XPS) was also used to follow modifications of the surface chemistry after fluid immersion.
Analysis was performed using a Gammadata Scienta SES 2002 X-ray photoelectron spectrometer under ultra high vacuum (p < 10?9 mbar). The monochromated Al K�� source (1486.6 eV) was operated at 420W (30 mA, 14 kV), with a nominal take-off angle of 90�� (i.e., photoelectrons ejection normal to the surface). The samples were outgassed into several ultra high vacuum chambers with isolated pumping system until transfer to the analysis chamber. No further cleaning process was made to avoid carbon contamination. During acquisition, the pass energy was set to 500 eV for survey spectrum with a step of 500 meV. The overall energetic resolution of the spectrometer can be estimated to 0.4 eV.
For quantification purpose, raw area of each photoelectron peaks was determined on survey spectrum using Shirley background and 30% Gaussian-Lorentzian shape with CasaXPS software (Casa Software Ltd.). Raw areas were further modified using classical sensitivity factors and transmission factor of the spectrometer leading to a chemical composition expressed in atomic percentage in the article. The analysis depth of XPS is approximately 8�C9 nm. XPS surface characterization was performed only for the T-TCP samples (one sample for each condition): the control T-TCP (pristine sample) and samples immersed in static or dynamic conditions, in complete and non-complete medium during 8 d (total 5 samples).
Calcium and phosphorous Brefeldin_A content in medium The concentration of calcium and phosphorus in the immersion medium after contact with the TCP and T-TCP tablets was evaluated at the end of each immersion time (1, 3 and 8 d) by colorimetric methods using a Calcium AS FS kit and Phosphorus UV FS kit purchased by Diasys Diagnostic Systems. Protein concentration in medium The concentration of total proteins in the immersion medium after contact with the TCP and T-TCP tablets was evaluated at the end of each immersion time (1, 3 and 8 d) by the Micro BCATM kit using the supplier instructions (Pierce). Protein concentration was obtained by comparison with BSA standards.