Fei nova nanosem 430

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×.

After the 30-day experiment, one sample of each specimen from each treatment group and one reference sample of each material were assessed under scanning electron microscopy (FEI NOVA NanoSEM 430, FEI Company, Hillsboro, OR, USA) to qualitatively evaluate surface topographical changes. Samples were sputter-coated with carbon to reduce charging effects during SEM analysis (10–15 mA, under a vacuum of 130 mTorr). The SEM was operated at 10 kV, spot 3.5.

The micro-architecture and mechanical properties of HA-Tyr gels in varying mechanical strength were examined by monitoring with scanning electron microscopy (SEM) as well as testing with the micro-mechanical tester. Gels were processed for SEM with freeze-dryer to remove the water, then sputtered with gold at 20 mA for 45 s, and examined under SEM (FEI 430 Nova NanoSEM, USA). In addition, compression test was performed on cylindrical hydrogels with 8-mm diameter and 5-mm heigth to determine the mechanical strength of 1, 3 and 6% HA-Tyr hydrogels. The analysis was performed at 2-mm/min loading rate and under a load of 5 N (Cellscale, Canada). Young’s moduli were calculated based on the stress/strain curves. All the measurements were performed in duplicate.