Fe 3

Metal ion stock solutions were prepared by adding chloride salts to a final concentration of 100 mM in dH₂O. Two different synthetic microbicidal mixtures (SMM3 and SMM16) were prepared by diluting metal ion stocks in dH₂O to the concentrations found in CB07-L [1] (link). Elemental chemistry of the CB leachates was previously measured by inductively coupled plasma mass spectrometry (ICP-MS) (described in Otto et al. [1] (link)), and ion concentrations from these ICP-MS measurements were used to prepare SMM3: Li (Sigma), 7.62 µM; Be(II) (Sigma), 0.80 µM; Na (Fisher), 171.61 µM; Mg(II) (Sigma), 773.11 µM; Al(III) (Sigma), 1079.6 µM; Ca(II) (Sigma), 6911.18 µM; Ti(IV) (Sigma), 0.01 µM; V(III) (Sigma), 0.49 µM; Mn(II) (Sigma), 99.31 µM; Fe(III) (Sigma), 229.88 µM; Co(II) (Sigma) 3.38 µM; Ni(II) (Sigma), 3.46 µM; Cu(II) (Sigma) 3.18 µM; Zn (Sigma) 14.52 µM [1] (link). The pH of the solution was adjusted to 3.5 with 1 M NaOH, and the solution was sterilized by passage through a 0.22 µM filter prior to further use. SMM16 was prepared in an identical manner as described above with the following metal ion concentrations: Fe(III), 200 µM; Co(II), 4 µM; Cu(II), 80 µM; Zn, 40 µM.

SPIO were prepared as described previously [15 (link)]. In brief, 0.1M Fe(III) (Sigma) and 0.2M Fe(II) (Sigma) aqueous solutions were prepared by dissolving FeCl₃ and FeCl₂, respectively. For production of Fe₃O₄ nanoparticles, 4 mL of the Fe(III) and 1 mL of the Fe(II) solutions were mixed at room temperature, and the pH was adjusted to 11 using 5M NaOH. After exposure to a magnet, the precipitates were washed with deionized water after which 3 g of organic acid was added to achieve complete coating of the particle surface. The precipitates were redispersed in deionized water after excess adherents were removed by centrifugation. Before labeling, 50 μg/mL of SPIO was coated by mixing 0.75 μg/mL poly-L-lysine (Sigma) into the culture medium at room temperature for 1 h to facilitate their endocytosis. MSCs were then seeded in a 6-well plate at a density of 4 × 10⁴/ well and cultured for 24 h. The MSCs were incubated in SPIO-containing medium for 24 h and thoroughly washed with PBS.