Titanium sheets were observed under scanning electron microscopy (FEI NOVA NanoSEM 430, FEI Company, Hillsboro, OR, USA) to identify cell attachments on their surface. For SEM analysis, one sample from each group was placed on a sterile 24-well plate and sterilized using ethanol 70% for 30 min. A quantity of 2 × 104 cells/well were cultivated on each titanium sheet at 37 °C in a 5% CO2 for 3 days.
The pre-osteoblasts that adhered to the samples were fixed in a solution of 3% glutaraldehyde (50 wt.% in H2O, CAS#111-30-8, Sigma-Aldrich, San Luis, MO, USA), 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich, San Luis, MO, USA) for 45 min. Samples were immersed for 10 min in a buffer solution of 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich, San Luis, MO, USA) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich, San Luis, MO, USA). Samples were then processed in serial ethanol dehydrations for 10 min each (30, 50, 70 and 100%) and dehydrated in hexamethyldisilazane (HDMS, CAS# 999-97-3, Sigma-Aldrich, San Luis, MO, USA).
Titanium sheets were sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, UK) with a thickness of 10 nm. The SEM was operated at 10 kV, spot 3.5 and images were made in 100×, 2000× and 10,000×.
The pre-osteoblasts that adhered to the samples were fixed in a solution of 3% glutaraldehyde (50 wt.% in H2O, CAS#111-30-8, Sigma-Aldrich, San Luis, MO, USA), 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich, San Luis, MO, USA) for 45 min. Samples were immersed for 10 min in a buffer solution of 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich, San Luis, MO, USA) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich, San Luis, MO, USA). Samples were then processed in serial ethanol dehydrations for 10 min each (30, 50, 70 and 100%) and dehydrated in hexamethyldisilazane (HDMS, CAS# 999-97-3, Sigma-Aldrich, San Luis, MO, USA).
Titanium sheets were sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, UK) with a thickness of 10 nm. The SEM was operated at 10 kV, spot 3.5 and images were made in 100×, 2000× and 10,000×.