Alizarin red staining solution

Tetraethylorthosilicate (TEOS, 99%) and chloride acid (HCl) (37%) were obtained from Alfa Aesar (Haverhill, MA, USA). Gelatin (type A porcine, gel strength 300) and 3-glycidoxypropyltrimethoxysilane (GPTMS, >98%) were purchased for Sigma Aldrich (St. Louis, MO, USA). Absolute ethanol (99.5%) was purchased from Panreac and acetid acid (Reagent Grade) was purchased from Scharlau (Barcelona, Spain). HOB^® human osteoblasts, fetal calf serum, osteoblast growing medium and osteoblast mineralization medium were purchased from Promocell (Heidelberg, Germany). Paraformaldehyde, PBS, Triton FITC conjugate were all purchased from Sigma, (St. Louis, MI, USA) and Vectashield^® (Vector Laboratories, Burlingame, CA, USA). Alizarin red staining solution was purchased from Fisher Scientific, (Waltham, MA, USA).

Mineralization capability was analyzed after 28 days under culture by Alizarin Red Staining Solution (Fisher Scientific, Waltham, MA, USA). Briefly, all cells were fixed with 4% paraformaldehyde for 1 h at room temperature, washed 3 times in phosphate-buffered saline, stained with 2% alizarin red solution, and again, rinsed 3 times in phosphate-buffered saline. Calcium deposits within cells and extracellular matrix were visualized under fluorescence microscope (magnification 10×). All experiments were performed in triplicate.