H2so4

D-Glucose, L-glutamic acid, NH₄Cl, K₂HPO₄, MgSO₄ × 7H₂O, CaCl₂ × 2H₂O, MnSO₄ × H₂O, FeCl₃ × 6H₂O, ZnSO₄ × 7H₂O, Na₂-EDTA, CuSO₄ × 5H₂O and CoCl₂ × 6H₂O were purchased from Carl Roth GmbH + Co., KG (Karlsruhe, Germany). LC/MS-grade ultra-pure water, HPLC-grade chloroform, acetic acid, H₂SO₄ and NH₄HCO₃ were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 10-Camphorsulfonic acid and tributylamine were purchased from SigmaAldrich (St. Louis, MO, United States).

PEEK plates (φ5.0 mm × 1 mm-thick; 450 G; VICTREX, UK) were prepared for each experiment. The surface of the plate was roughened using a sandblasting machine (BUEHLER, IL, USA) with silicon carbide paper (#600; SANKYO RIKAGAKU, Japan), followed by ultrasonic washes with pure water and ethanol. The PEEK surface was then immersed into 98% concentrated H₂SO₄ (WAKO CHEMICAL Japan) for various periods of time (1, 3, 5, 10, and 15 min) to produce a binding surface. After dry heat treatment at 110 °C for 30 min, a soft solution process was applied consisting of simulated body fluid (SBF), urea, and urease. Specifically, urea in SBF was adjusted to 2.0 mol/dm³, and applied to the PEEK surface, followed by a 1.0 mass % urease solution, at 0.0534 cm³ per 5 cm³ of PEEK surface. The material was heated at 36.5 °C for 24 h, followed by its reimmersion in SBF. The material was then placed at 50 °C, and the SBF was exchanged every 48 h, for a total of 3 times. By this treatment, HAp film was produced on the surface of the PEEK substrate. The substrate was then immersed in IP6 solution (1000 ppm) at 50 °C for 24 h, followed by 10 min in AgNO₃ solution (5 mmol·dm⁻³) at 25 °C, according to our previous protocol^{14 (link)}.

B cell-mediated humoral responses were measured as virion-specific immunoglobulin production by ELISA, as previously described51 (link). Briefly, 96-well ELISA plates (Corning, Corning, NY) were coated with ultrasonicated influenza virion (A/California/07/2009 strain) at 5 × 10⁶ PFU/mL in a carbonate buffer (pH 9.6) and incubated overnight at 4 °C. Plates were then washed with PBS containing 0.05% Tween 20 (Wako). Serum and BALF collected from mice at day 7 after the secondary infection were serially diluted with PBS/Tween 20 containing 5% skim milk, applied onto the virion-coated plates, and incubated for 2 h at room temperature. After washing, goat anti-mouse total IgG or IgA conjugated to horseradish peroxidase (Jackson Immunoresearch, Baltimore Pike, PA) was applied and incubated for 2 h at room temperature. After washing, the plates were stained with a TMB Substrate Set (Biolegend). The reaction was terminated with 1 M H₂SO₄ (Wako) and the absorbance was measured.

In order to form pores on the surface of the Sample N, the Sample N was soaked in 40 cm³ of 95 wt % H₂SO₄ (FUJIFILM Wako Pure Chemical) for 4 s in total. Thus, treated PEEKs were washed ultrasonically in distilled water for 10 min. The obtained samples are denoted as ‘Sample S’ hereafter.