Calcium nitrate tetrahydrate

Mesoporous bioactive glass nanoparticles (MBN) were synthesized with a modified method from Lee et al. [36 (link)] and El-Fiqi et al. [37 (link)]. In 150 mL distilled water, 2-ethoxyethanol (Sigma-Aldrich, St. Louis, MO, USA), 2 mL aqueous ammonia (Samchun, Pyeongtaek, South Korea), 1.4 g calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O) (Sigma-Aldrich), and 20 mL ethanol (Samchun, Pyeongtaek, South Korea) were combined; then, 1 g hexadecyltrimethylammonium bromide (CTAB) (Sigma-Aldrich) was added, and the mixture was stirred for 30 min at room temperature. After adding 5 mL of tetraethyl orthosilicate (TEOS) (Sigma-Aldrich), 4 h of vigorous stirring was performed. The molar ratio (mol %) of the resulting CaO:SiO₂ was calculated to be 15:85. A white precipitate was obtained, which was washed with ethanol and dried for 24 h at 60 °C. To remove CTAB, calcination was performed for 5 h at a heating rate of 1 °C·min⁻¹ at 600 °C.

Ti6Al7Nb alloy was used as the substrate for the synthesis of nHA and GNS containing coatings in this study. The chemical composition of the substrate is presented in Table 1. The alloy plates supplied with 50 × 50 × 0.4 cm and then, cut by using a water jet machine with dimensions of 2.5 × 2.5 cm². The cut surfaces were ground with sandpapers step by step from 240 to 1200 to remove the cutting traces formed on their surfaces. Then, the surfaces were ultrasonically cleaned with acetone, ethanol and deionized water, respectively.Table 1

Chemical composition of Ti6Al7Nb alloy (in wt.%)

Al	Nb	Fe	O	N	Ti
6.12	7.07	0.12	0.18	0.01	bal.

Disodium edetate (C₁₀H₁₄N₂Na₂O₈·2H₂O), sulfuric acid (H₂SO₄, 98%), hydrogen peroxide (H₂O₂, 30% aq.), calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O), di-ammonium hydrogen phosphate ((NH₄)₂HPO₄) and ammonium hydroxide (NH₄OH 28% aq.) were purchased commercially from Sigma-Aldrich. Graphene nanosheets (GNS) were purchased from Nanografi Company with 99% pureness. All chemicals were used analytically and without further purification.

All the chemicals were used as received from the manufacturer. For the preparation of the HA materials, calcium nitrate tetrahydrate (Ca(NO₃)·4H₂O, AR), triethyl phosphate (P(OC₂H₅)₃, AR), diammonium hydrogen phosphate ((NH₄)₂HPO₄, AR), Pluronic F127, polyvinyl alcohol (PVA), and 10× concentrated phosphate buffered saline (10× PBS) all came from Sigma-Aldrich, while anhydrous ethanol was from Merck (Darmstadt, Germany). Protein adsorption and cell culture studies required bovine serum albumin (BSA), vitronectin, BCA^TM Protein Assay Kit, hexamethyldisilane (HMDS), and sodium dodecyl sulphate (SDS, AR) all from Sigma-Aldrich; Minimum Essential Media (MEM), Glutamax, Non-Essential Amino Acids (NEAA), Antibiotic-Antimycotic solution (Anti-Anti), and TrypLE Express were purchased from Invitrogen; MTS reagent from Promega, and Fetal Bovine Serum (FBS) from SAFC Biosciences (Hampshire, England). The water used in all experiments was passed through a Millipore (Boston, MA, USA) Milli Q ultrapure water purification system and had a resistivity higher than 18.2 MΩcm.

Silver nitrate (Sigma-Aldrich, 99.99%) was employed as the precursor for silver dopant. The main precursors for HA synthesis include ammonium phosphate mono-basic (Sigma-Aldrich, 98%), and Calcium nitrate tetrahydrate (Sigma-Aldrich, 99%). Crystal violet (Sigam-Aldrich, 99%) was employed as contaminant. All chemicals were used as received without further treatment.

Sodium alginate, calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O), ammonium phosphate dibasic ((NH₄)₂HPO₄), caffeic acid, chlorogenic acid, cisplatin, ammonium hydroxide solution (NH₄OH), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma-Aldrich Co. and were used without further purification. The human osteosarcoma cell line (MG-63) was obtained from the American Type Culture Collection (ATCC, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, and Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4) were obtained from Gibco BRL (USA). Slowfade gold antifade reagent and Live/Dead Viability/Cytotoxicity assay kit were purchased from Molecular probes (USA). Other reagents and solvents were commercially available and were used as received.