Ammonium phosphate dibasic nh4 2hpo4

All materials used in the synthesis of GelMA (Type A gelatin, Methacrylic anhydride (MA), dialysis tubing, high-retention seamless cellulose tubing (12–14 kDA MWCO, 40 mm diameter), Dulbecco’s phosphate-buffered saline (DPBS) Dialysis tubing closures), fabrication of hydrogel (Triethanolamine (TEA), Eosin Y disodium salt, N-Vinylcaprolactam (VC)), fabrication of biphasic calcium phosphate nanoparticles (BCP-NP) (calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O, and ammonium phosphate dibasic ((NH₄)2HPO₄) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Moreover, α-MEM (Minimum Essential Medium Eagle-α), 10% fetal bovine serum (FBS), and antibiotics (penicillin/streptomycin) used for the cell experiment were purchased from Gibco BRL (Invitrogen Co., Carlsbad, CA, USA). Cell proliferation and activity were evaluated, using CCKi-8 (Enzo Life Science Inc., New York, NY, USA) and a TRACP & ALP assay kit (TakaRa, Kyoto City, Kyoto Prefecture, Japan).

Calcium nitrate tetrahydrate (Ca(NO₃)₂.4H₂O) (99%, ACS reagent), ammonium phosphate dibasic ((NH₄)₂HPO₄) (≥98.0%, reagent grade) and agar (for microbiology) were acquired from Sigma-Aldrich (Sigma-Aldrich, UK). All mixtures and solutions were prepared with H₂O with a recorded resistivity of 15–18 MΩ.cm (at 25 °C and 1 atm) from a Milli-Q^® Integral 3 water purification system equipped with Q-Gard^® and Quantum^® cartridges (Merck Millipore, USA). Calcium-loaded hydrogel was prepared by heating a mixture of Ca(NO₃)₂.4H₂O ([Ca²⁺  = 1 M) and 5 w/v% agar to 80–90 °C whilst stirring at 250–500 rpm on a magnetic stirrer hot plate. Using a syringe, 15 mL of warm hydrogel mixture was cast evenly into the chemical garden liquid exchange unit by hand. The liquid exchange unit and its contents were then stored at 4 °C overnight to complete gelation before returning to room temperature. Self-assembly of tubular calcium phosphate frameworks was initiated by the addition of 85 mL phosphate solution prepared with (NH₄)₂HPO₄ ([HPO₄²⁻] = 1 M).

HA was produced using the sol-gel method. Here, 0.5 M calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O) (Sigma-Aldrich, Saint Louis, MO, USA) and 0.3 M ammonium phosphate dibasic ((NH₄)₂·HPO₄) (Sigma-Aldrich, Saint Louis, MO, USA) were used as reagents, and the Ca:P ratio was set to 1.67. To produce HA, a 0.3 M ammonium phosphate dibasic solution was added to a 0.5 M calcium nitrate tetrahydrate solution at a 1:1 ratio. Here, an ammonia solution (SHOWA, Urawa, Japan) was added to raise the pH of the solution to at least 10. The solutions were stirred for 3 h. After, the solution aged for 24 h at 25 °C in a Forced Convection Oven (OF-12GW, JEIO TECH, Deajeon, Korea) and was dried to obtain the HA powder.