Silver nitrate

For cytological stainings, osteoblast cultures were rinsed with PBS and fixed with 3.7% formalin for 10 min. After dH₂O washes formalin-fixed wells were stained for ALP with Naphthol AS MX-PO₄ (Sigma, USA) dissolved in DMF (Sigma USA) mixed with Fast Blue RR salt (Sigma, USA) in 0.1 M Tris-HCl (pH 8.3). ALP stained wells were stained for von Kossa with 2.5% Silver nitrate (Fisher Scientific, UK) for 30 min exposed to direct light, and washed with dH₂O for three times. ALP and von Kossa stained six-well plates were scanned using a flatbed scanner with a transparency adaptor (HP ScanJet 5370 C) and saved as 24-bit color images in TIFF format. Transparency exposure adjustments were maintained constant to create images of equal intensity. Positively stained areas were quantified using Imaging Software ImageJ. RGB images were split into three 8-bit grayscale images containing the red, green and blue components. Threshold and Region of Interest (ROI) were adjusted and kept the same to maintain standard measuring conditions.

Polyvinyl alcohol (Sigma Aldrich Chemical Company, St. Louis, MO, USA) was purchased as a fully hydrolyzed powder with an average molecular weight of 85,000 g/mol. For the hybrid method, CA membranes were commercially purchased from Fisher Scientific Company (Waltham, MA, USA) with a symmetrical pore size of 0.45 µm and a diameter of 47mm. The tridentate chelator, iminodiacetic acid (IDA), and 50% (w/w) GA were also purchased from Sigma-Aldrich Chemical Company. Bovine serum albumin, sulfuric acid, copper sulfate, silver nitrate, ethylenediaminetetraacetic acid (EDTA), acetic acid, and sodium bicarbonate were purchased from Fisher Scientific Company. All materials were used without further purification.

Potassium tetrachloroplatinate(II), l-alanine, trimethylsilyl propionate (TSP), and all nucleosides were purchased from Sigma-Aldrich (St. Louis, MO, USA). The l-ornithine and cis-diamminedichloridoplatinum(II) were from Alfa Aesar (Haverhill, MA, USA). Deuterium oxide (D₂O) was from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Silver nitrate, acetonitrile, methanol, and ammonium acetate were obtained from Fisher Chemical (Hampton, NH, USA). Sodium phosphate, monobasic and dibasic, was obtained from EMD Millipore (Kankakee, IL, USA).

Silver nitrate, which is less soluble in compounds with ether groups and more soluble in aqueous ammonia, was purchased from Fisher Scientific Co. in crystalline form, with more than 98% purity and with a melting point of greater than 200 °C.

Von Kossa staining was performed as previously reported^{70 (link)}. In brief, the cells were fixed in 4% formaldehyde (Fisher Scientific, Fair Lawn, NJ, USA) after 7- and 28-days culture periods and incubated with 2% silver nitrate (Fisher Scientific) in dark for 10 min and then exposed to bright light for 15 min. The plates were then washed with distilled water and dehydrated in 100% ethanol. The samples were then examined by light microscopy^{71 (link)}.