Branson 5200

The mobile phase was composed of MeOH and water (MeOH/H₂O) at a ratio of 50:50 (v/v) to which trifluoroacetic acid (0.1% v/v) was added. The mobile phase was then degassed using a Branson 5200 ultrasonic bath (Branson Ultrasonics, Danbury, CT).

Titanium discs (12.00 ± 0.04 mm in diameter and 2.99 ± 0.02 mm in thickness) made of commercially pure titanium were prepared as described previously^{6 (link), 7 (link)}. Briefly, a grinder–polisher (Ecomet 3, Buehler, Lake Bluff, IL, USA) was used to polish the discs with an ascending series of grinding papers (CarbiMet, Buehler) with grit numbers of 120, 240, 400, and 600. Then, the titanium discs were sandblasted (EWL Type 5423, KaVo) using a 50-μm white aluminum oxide blasting compound (Ivoclar Vivadent, Liechtenstein). Then, the sandblasted discs were sonicated (Branson 5200, Branson Ultrasonics, Danbury, CT) twice in ultrapure water for 5 min to eliminate residues of the aluminum oxide blasting compound. The discs were then soaked in acetone (Sigma-Aldrich, St. Louis, MO) for 15 min to remove any contaminants from their surface. Then they were acid etched with 30% nitric acid (ThermoFisher Scientific, Fair Lawn, NJ) for 30 min. Finally, the discs were rinsed with ultrapure water and stored in 70% ethanol at room temperature.

Titanium discs made of commercially pure titanium were prepared as described previously (Atluri et al., 2017 (link)). Briefly, discs were sanded with a variable speed grinder-polisher (Ecomet 3, Buehler, Lake Bluff, IL, USA) using grinding papers (CarbiMet, Buehler) ascending to grit number 600. The titanium discs were then sandblasted (EWL Type 5423, KaVo, Germany) using 50 μm white aluminum oxide blasting compound (Ivoclar Vivadent, Liechtenstein). The sandblasted discs were then sonicated (Branson 5200, Branson Ultrasonics, Danbury, CT, USA) twice in ultrapure water for 5 minutes each to remove any remnants of blasting compound. The discs were degreased with acetone for 15 minutes, then acid etched with 30% nitric acid for 30 minutes. The discs were then rinsed with ultrapure water and stored in 70% ethanol. Sandblasting followed by acid etching is a common method used in the fabrication of dental implants with roughened titanium surface and has been used extensively in implantology research to create titanium surfaces that mimic commercial dental implant surface for in vitro evaluations (Bowers et al., 1992 (link); Ko et al., 2010 (link); Schneider et al., 2004 (link)).