Silver nitrate

Corning Osteo Assay Surface multiple-well plates (Corning, NY, USA) were used to directly assess the osteoblastic activity in vitro as described previously [30] (link), [31] (link). In brief, the MSCs were seeded at 2×10⁴/well (1×10⁴/cm²) in a Corning Osteo Surface plate (Corning, NY, USA) with the osteogenic medium. The osteogenic medium was refreshed every 3 days. The formed bone nodules were determined on Day 18 after osteogenic differentiation commenced and were measured using von Kossa staining. Briefly, cells were washed with PBS 3 times and fixed with 4% paraformaldehyde in PBS for 45 minutes at room temperature. After washing with deionized water, the cells were stained with 5% silver nitrate (Sigma-Aldrich, MO, USA) for another 45 minutes at room temperature under a bright light. To stop the silver nitrate reaction, the cells were washed with water and treated with a 5% solution of sodium thiosulfate (Sigma-Aldrich, MO, USA). Following another water wash and air drying, the nodules were visualized as dark staining patches under a light microscope. The area of von Kossa-positive nodules was determined using the public Image J software (developed by NIH, rsb.info.nih.gov/ij/) and the total areas (µm²) of the von Kossa-positive nodules were calculated.

Ethephon (2-chloroethylphosphonic acid; 2SL, Makhteshim Agan of North America, Inc., Raleigh, NC 27604, USA) solution was prepared with 500 ppm (w/v) ethephon. Silver thiosulfate (STS) was prepared as follows: A 0.1 mol/L solution of silver nitrate (Sigma-Aldrich, St. Louis, MO, USA) was slowly mixed into a 0.1 mol/L sodium thiosulfate (Sigma-Aldrich) at a 1:4 silver nitrate to sodium thiosulfate ratio by volume. The resulting STS stock solution, with 20 mmol Ag⁺/L, was diluted with reverse osmosis (RO) purified water to the desired concentrations described below and in figure and table legends, where the reported mM concentration of STS refers to the Ag⁺ concentration contained therein. Each solution contained 0.1% (v/v) Tween 20 (Sigma-Aldrich, PO Box 14508 St. Louis, MO 63178, USA). For all STS experiments except the localization and timing experiment (described below), a 100 mL treatment of each was applied by spraying all leaves using a 1.5-gallon Solo® 450 series sprayer (Solo, 5100 Chestnut Avenue Newport News, VA 23605) with a 0.14 MPa (21 psi) constant flow valve (item no. 163124, Gempler P.O. Box 5175, Janesville, WI, USA).

Silver NPs were synthesized by the reduction of silver nitrate (≥99.0%, Sigma-Aldrich) following a previous report^{32 (link)} with appropriate modification. Typically, 240 μL of 0.1 M l-ascorbic acid (99%, Sigma-Aldrich) and 3.3 mL of 0.1 M sodium citrate (anhydrous, ≥99.5%, Sigma-Aldrich) were injected into a flask containing 240 mL of boiled deionized water under continuous stirring. After heating for one minute, 6 mL of 0.01 wt% polyvinylpyrrolidone (PVP, average molecular weight 360 000, Sigma-Aldrich) and 800 μL of 0.1 M aqueous silver nitrate were added to the flask sequentially. The heating and stirring were stopped after 5 minutes and a uniform orange solution was obtained. The solution was then cooled down to room temperature and stored in the fridge.

Tetraethylorthosilicate (TEOS),
cetyltrimethylammonium bromide (CTAB), sodium hydroxide (NaOH), chloroauric
Acid (HAuCl₄), silver nitrate (AgNO₃), hydrochloric
acid (HCl), sodium citrate dibasic trihydrate, gold chloride trihydrate
(HAuCl₄·3H₂O), sodium citrate tribasic
dihydrate, silver nitrate (AgNO₃), l-ascorbic
acid (AA), hydrogen peroxide solution (H₂O₂,
30 wt %), (3-mercaptopropyl)trimethoxysilane (MPTMS, 95%), phosphate-buffered
saline (PBS, pH 7.4), DMEM tissue culture medium, and bovine serum
albumin (BSA) were purchased from Sigma-Aldrich, UK. Hydrochloric
acid (HCl, 37%), sulfuric acid (H₂SO₄, 96%),
acetone, and 2-propanol were obtained from VWR International, UK.
Deionized (DI) water was purified using the Millipore Milli-Q gradient
system (>18.2 MΩ).
Peptides (sequences H3: KELLTRELPSFLGKRT,
H6: NEFFEGFPDKQPRKK) were synthesized as tetramers composed of four
monomers coupled to a lysine backbone (Schafer-N, Denmark). Tetramerization
was previously found to be necessary for the neuritogenic activity
of S100A4.^{23 (link)}