Calcium nitrate tetrahydrate

Calcium nitrate tetrahydrate, (Ca(NO₃)₂.4H₂O, 99%, Alfa Aesar), ammonium molybdate (H₈MoN₂O₄, 99.99%, Alfa Aesar, Ward Hill, MA, USA), europium(III) nitrate hydrate (Eu(NO₃)₃ .xH₂O, 99.99%, Sigma Aldrich, St. Louis, MO, USA), oleic acid (Alfa Aesar), 1-octadecene (95%, Alfa Aesar), NaOH pellet (Merck, Kenilworth, NJ, USA), hydrochloric acid (HCl, Sigma-Aldrich), HS-PEG-COOH (MW = 5000, NANOCS, New York, NY, USA), mPEG-SH (MW = 5000, NANOCS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (C₈H₁₇N₃HCl, MW = 191.7 g mol^–1, Sigma-Aldrich) (EDC), N-hydroxysuccinimide (C₄H₅NO₃, Sigma-Aldrich, MW = 115.09 g mol^–1) (NHS), 4-mercaptobenzoic acid (MBA) (Sigma-Aldrich), anti-human epidermal growth factor receptor (EGFR) antibody (Thermo Fisher Scientific, Waltham, MA, USA), and phosphate buffered saline (1×) (PBS) (Thermo Fisher Scientific) were used for HNP synthesis. Human (Homo sapiens) lung carcinoma (A549 cells) (ATCC, USA), mouse hepatocyte cells (AML12, normal hepatocyte from liver tissue), ethylenediaminetetraacetic acid (EDTA) stabilized human whole blood was freshly obtained from Innovative Research (Novi, MI, USA), 0.5% trypsin-EDTA solution (Life Technologies), LIVE/DEAD viability/cytotoxicity assay kit (Life Technologies), Earle’s balanced salt solution (EBSS) (Life Technologies) and PBS were used for cell experiments.

Pluronic P123 (EO₂₀:PO₇₀:EO₂₀, M_n = 5800, Sigma-Aldrich),
tetraethyl orthosilicate (TEOS; reagent grade 98%, Sigma-Aldrich),
HCl (37–38%, J.T. Baker), magnesium acetate tetrahydrate (MgAc₂·4H₂O; ACS reagent grade 99%, Sigma-Aldrich),
aluminum isopropoxide (Al(−OⁱPr)₃; ≥98%, Sigma-Aldrich), calcium nitrate tetrahydrate
(Ca(NO₃)₂·4H₂O; 99%, Alfa Aesar),
titanium(IV) isopropoxide (Ti(−OⁱPr)₄; 97%, Sigma-Aldrich), zirconyl chloride octahydrate
(ZrOCl₂·8H₂O; ≥98%, Acros Organics),
sodium chloride (NaCl; ACS reagent grade ≥99.0%, Sigma-Aldrich),
and cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6H₂O; ACS reagent grade 99.5%, Sigma-Aldrich) were
used as purchased.

PLLA opaque pallets having an average molecular mass of 100,000 g/mol suitable for bone-tissue engineering were purchased from Polysciences Inc., Philadelphia, PA, USA. COC TOPAS^® 8007 amorphous polymer was obtained from Ticona, Sulzbach, Germany. Calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O), reagent grade, molecular weight 236.15 g/mol assay (complexometric assay 99–103%), di-ammonium hydrogen phosphate ((NH₄)₂HPO₄), reagent grade, molecular weight 132.06 g/mol (98%), and ammonia solution 35% NH₃ sol, certified AR for analysis, d = 0.88, Fisher Chemicals, Hampton, NH, USA were acquired and used as such. Dulbecco’s Modified Eagle’s Medium (DMEM), Trypsin-EDTA (0.05%), phenol red, LIVE/DEAD^® Viability/Cytotoxicity Kit (mammalian cells) and fetal bovine serum (FBS) were procured from (Gibco) Thermo Fisher Scientific Inc, Waltham, MA, USA. Chloroform, research grade (99% pure), penicillin-streptomycin (10,000 U/mL), Triton X-100, rhodamine phalloidin (Alexa Fluor 594-labeled, Invitrogen™, Waltham, MA, USA), 4% paraformaldehyde (PFA), bovine serum albumin (BSA), 4′,6-diamidino-2-phenylindole (DAPI), and phosphate buffered saline (PBS) were procured from Sigma-Aldrich, Burlington, MA, USA. Two frozen cell lines: frozen MC3T3-E1 (pre-osteoblast cell lines) and BMSC (bone marrow-derived mesenchymal stem cells), were obtained by the courtesy of ATCC, Manassas, VA, USA.

Calcium nitrate tetrahydrate (Fisher, Hampton, NH, USA), ammonium phosphate (Fisher, USA), ammonium hydroxide (Sigma Aldrich, Oakville, ON, USA), hydroxyapatite (Sigma Aldrich, Oakville, ON, Canada), atelo-collagen solution (Advanced BioMatrix, Carlsbad, CA, USA), sodium hydroxide (Fisher, USA), and sodium alginate (DuPont, Wilmington, DE, USA) were utilized for ink preparation. Calcium chloride (Fisher, Schwerte, Germany) and glutaraldehyde (Sigma-Aldrich, Schnelldorf, Germany) were used as crosslinking agents. Thymol (Fisher, USA), ethanol (Fisher, USA), and citric acid (Sigma Aldrich, USA) for dentin disk preparation and polystyrene microspheres (Sigma Aldrich, USA) for the occlusion test were utilized. For cell study, bone marrow-derived stem cells (Lonza, Bend, OR, USA), high glucose DMEM (Gibco, Thermo Fisher, Ottawa, ON, Canada), fetal bovine serum (Gibco, Canada), 1% penicillin–streptomycin (Gibco, Canada), ascorbic acid (Sigma Aldrich, Oakville, ON), dexamethasone (Thermo Fisher, Waltham, MA, USA), and β-glycerophosphate (Sigma Aldrich, Oakville, ON, Canada) were used.

85∶15 poly(lactide-co-glycolide) (PLGA) with a MW of ∼94,000 Da was purchased from Lakeshore Biomaterials (Birmingham, AL). Methylene chloride, calcium nitrate tetrahydrate, ammonium hydrogen phosphate, and polyvinyl alcohol (PVA) (MW ∼70,000 Da) were acquired from Fisher Scientific (Pittsburgh, PA). Ammonium hydroxide, collagenase type I, ascorbic acid, β-glycerophosphate, vitamin D₃, and cetylpyridinium chloride (CPC) were obtained from Sigma-Aldrich (St. Louis, MO). Dulbecco's Phosphate-Buffered Saline (PBS) and 0.05% trypsin-EDTA solution were purchased from Gibco (Grand Island, NY). Dulbecco's Modified Eagle Medium (DMEM), penicillin-streptomycin solution (Pen-Strep), and fetal bovine serum (FBS) were procured from Invitrogen Corporation (Carlsbad, CA). Triton X-100 was bought from Bio-Rad Laboratories, Inc. (Hercules, CA). Recombinant human bone morphogenetic protein-2 (rhBMP-2) was purchased from GenScript Corporation (Piscataway, NJ).