Synthesis and Characterization of Calcium Phosphate Particles

CPP were synthesized as previously reported^{6 (link),21 (link)}. Briefly, a CPP solution containing 1 mg/mL bovine fetuin-A (Sigma-Aldrich, St. Louis, MO, USA), 10 mM CaCl₂, 6 mM Na₂HPO₄, 140 mM NaCl, and 50 mM Tris–HCL (pH 7.4) was gently shaken and incubated at 37 °C for at least 24 h. Synthetic CPP were separated using a spin-filtered membrane cartridge with a 300 kDa cut-off (Sartorius AG, Göttingen, Germany). CPP concentrations were determined by bicinchoninic acid (BCA) assay as a protein level. The ionic calcium content of secondary CPP was also measured by Calcium Assay Kit (Cat No. ab102505, abcam, Cambridge, UK). One hundred μg/mL of secondary CPP as a protein concentration included 45 μg/mL of ionic calcium (Supplementary Fig. 8). Synthetic secondary CPP were used for experiments from 24 to 168 h after the first mixture of the solution.