Cas 57 50 1

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×.

Flies were fasted for 24 h prior to preparation for proboscis extension reflex (PER) assay. Flies anesthetized using CO₂ were sorted out and size matched females were adhered by their dorsal side of their thorax to a microscopy slide by using nail polish (Cat#72180, Electron Microscopy Science) as described previously^{2 (link),38 (link)}. After varying lengths of recovery time, PER was measured as follows: flies were water satiated and were used for experiments only after not responding to water three consecutive times. Flies which continued responding to water after 5-min were excluded from the experiment. Then, 10 mM or 100 mM fructose (CAS#57-48-7, Sigma Aldrich) was applied briefly (1 s) to the tarsi and the proboscis extension was recorded (only binomial yes/no response was recorded). Each tastant was presented five consecutive times with water used for tarsi washing and satiation (as needed) between each trial. 1 M sucrose (CAS#57-50-1, Sigma Aldrich) was applied at the end of each session to check for the responsiveness of each fly. Flies that did not respond to sucrose at the end were excluded. An index of PER response was calculated as a percentage of proboscis extensions out of the total number of tastant presentations per fly and per group.

Titanium sheets were observed under scanning electron microscopy using a MAICE system (JEOL JSM-6400 Scanning Electron Microscope, JEOL LTD, Tokyo, Japan) to identify cell attachments on their surface. The osteoblasts that adhered to the samples were fixed in a solution of 3% glutaraldehyde (50 wt.% in H₂O, CAS#111-30-8, Sigma-Aldrich), 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich), and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich) for 45 min. Samples were soaked for 10 min in a buffer solution of 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich) and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich). Sample surfaces and cells were processed in serial ethanol dehydrations for 10 min each and dehydrated in hexamethyldisilazane (HDMS, CAS# 999-97-3, Sigma-Aldrich) before stored in a desiccator until SEM imaging. Titanium sheets were then sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, UK) with a thickness of 10 nm (10–15 mA, under a vacuum of 130 mTorr). After this, the SEM was operated at 5 kV, spot 3 to 6.
The quantitative data were shown as the means ± standard deviations. The statistical differences were calculated using one-way ANOVA and Tukey’s test (GraphPad Prism 9 Software, Inc, San Diego, CA, USA). A p-value of ≤0.05 was considered statistically significant.