Nh4 2hpo4

Type I COL was extracted from calf skin by pepsin digestion, and dissolved in 0.3 M acetic acid solution at 4°C to obtain the COL acidic solution (8 mg/ml) for this study [38 ]. NaOH and acetic acid were purchased from Kelong Chemical Co. (Chengdu, China). CaCl₂, Ca(NO₃)₂, (NH₄)₂HPO₄ and NaH₂PO₄·2H₂O were purchased from Sigma-Aldrich Co. LLC. (MO, USA). Phosphate buffer saline (PBS) was provided by Solarbio Life Sciences (Beijing, China). α-Minimum Essential Medium (α-MEM, Hyclone), fetal bovine serum (Gibco), trypsin (Hyclone), penlcillin-streptomycin solution (Hyclone), Dex (Sigma), β-glycerophosphate (Sigma) and L-ascorbic acid (Sigma) were all used in cell culture experiments. Fluorescein diacetate (FDA), tetramethylrhodamine (TRITC)-phalloidin and Hoechst 33342 were purchased from Sigma-Aldrich (St Louis, MO). The DNA primers of β-actin, ALP, COL-I, OCN, RUNX-2 and OSX were synthesized by Tiantai Life Science (Chengdu, China).

The chemicals used to accomplish the experiments are as follows: (NH₄)₂HPO₄ (98%) from Sigma–Aldrich, Ca(NO₃)₂*4H₂O (99%), NH₄OH (25% solution), Ce(NO₃)₃*6H₂O (99%), Mg(NO₃)₂*6H₂O (99%) from Fluka—Honeywell Research Chemicals, and ultrapure water.

Bacteria were precultured overnight. A bacterial suspension was inoculated in minimal medium peptone (MMP) medium [2.5 g/l K₂HPO₄ (Baker, Deventer, Holland), 1.0 g/l (NH₄)₂HPO₄ (SIGMA, Steinheim, Germany), 0.2 g/l MgSO₄^∗7H₂O (Carlo Erba, Rodano (MI), Italy), 0.5% Bacto Peptone (Biolife, Milano, Italy)] and cultured at 28°C with shaking at 120 rpm up to Optical Density (OD) of 0.5. A spectrophotometer Ultrospec 3100 pro (Amersham pharmacia biotech) was used for the OD₆₀₀ measurement. For the degradation assay, bacteria were inoculated at 2% in a final volume of 5 ml of MMP medium supplemented with OTA (1 μg/ml) and grown for 144 h. Bacterial growth and cell viability were monitored by measuring the OD₆₀₀ and plate counting for each time point (0, 3, 6, 12, 24, 48, 72, 96, 144 h). For the plate counting, serial dilutions of the bacterial suspensions were prepared in 0.85% NaCl (AppliChem, Darmstatd, Germany), 0.1% Tween (Fisher Scientific, Bridgewater, NJ, USA) H₂O sterile solution and spread-plated on pre-poured PCA plates. Plates were incubated at 28°C overnight and CFU were counted. Each experiment was performed in triplicate. One milliliter of the culture was centrifuged and the supernatant was used for the chemical analysis.

The main substrates used for fabrication of the Ca₁₀(PO₄)₆(OH)₂ nanoparticles (nHAP) using a co-precipitation technique were Ca(NO₃)₂·4H₂O (99.98% Alfa Aesar, Karlsruhe, Germany) and (NH₄)₂HPO₄ (99.99% Sigma Aldrich, Saint Louis, MO, USA) as well as NH₄OH (99% Avantor Performance Materials Poland S.A., Gliwice, Poland) for pH control.
In this method, stoichiometric amounts of Ca(NO₃)₂·4H₂O and (NH₄)₂HPO₄ were dissolved in MQ-water separately. Subsequently, the prepared solutions were mixed. As a result, there was fast precipitation of the by-product. The solution pH was adjusted to 10 with NH₄OH under constant and vigorous stirring at 90 °C for 2 h. Finally, the by-product was dried for 24 h at 90 °C and thermally treated at the temperature range of 800–1000 °C for 3 h, resulting in formation of white, fine-grained powders.

HAP with a high surface area was synthesized using a hydrothermal method based on a previous report by Jiang et al.30 . In a typical synthesis procedure, 2 mmol of CaCl₂˙2H₂O (Sigma-Aldrich, ⩾99%) and 1 g of poly-L-aspartic acid sodium salt (Sigma-Aldrich, mol. wt. 5,000–15,000) was dissolved in 20 ml of deionized water under 30 min of stirring (solution 1). Then, 1.2 mmol of (NH₄)₂HPO₄ (Sigma-Aldrich, ⩾98%) was dissolved in 15 ml of deionized water under vigorous stirring for 30 min (solution 2). After stirring, solution 2 was poured into solution 1. The mixed solution was sealed in a 50-ml Teflon liner, placed in a stainless steel autoclave and maintained at 200 °C for 12 h. The precipitated HAP was washed with distilled water and ethanol. After washing, the obtained HAP powder was dried in a freeze dryer for 12 h.