Glycerol phosphate disodium salt hydrate

All salts and solvents, unless otherwise stated, were obtained from Fisher Scientific (UK). Sodium phosphate monobasic monohydrate, low viscosity chitosan from shrimp, glycerol phosphate disodium salt hydrate, tris(hydroxymethyl)amino-methane [(HOCH₂)₃CNH₂], albumin from bovine serum (BSA), albumin from chicken egg white (OVA) and QuantiPro™ Bicinchoninic Acid Assay Kit were obtained from Sigma-Aldrich (UK). l(+)-lactic acid 90 % solution in water was obtained from Acros Organics (USA). Dialysis membranes (size 10, MWCO 12–14 kDa) were obtained from Medicell International Ltd (UK). MWCTs and SWCTs were purchased from Cheap Tubes Inc. (USA). The MWCTs used in the study were 8–15 nm in diameter and 10–50 µm in length. The SWCTs used were 1–4 nm in diameter and 5–30 µm in length. Di-potassium hydrogen orthophosphate anhydrous (K₂HPO₄) was acquired from British Drug Houses (UK) and troclosene sodium dehydrate from Guest Medical (UK). Water used in all experiments was purified water. NOSC and chitosan modified hydroxyapatite (HACS) were prepared as described previously [12 (link), 21 (link)].

All solutions were prepared using ultrapure water (MilliQ, Millipore, France). For use in synthesis by enzymatic catalysis, alkaline phosphatase isolated from bovine intestinal mucosa (ALP, lyophilized powder, ≥10 DEA units/mg solid), glycerol phosphate disodium salt hydrate (S, isomeric mixture, typically 50% β-isomer and 50% rac-α-isomer), calcium chloride (CaCl₂, anhydrous, ≥99%), and sodium fluoride (NaF, BioXtra, ≥99%) were all sourced from Sigma-Aldrich (France). All solutions were prepared in 10 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, pH 7.4, made from Trizma^® base (minimum 99.9% titration) and 1 N hydrochloric acid (HCl). For measurement of enzymatic activity, 4-nitrophenol (pnp, ReagentPlus^® ≥ 99%) and 4-nitrophenyl phosphate disodium salt hexahydrate (pnpp, ≥99%) were sourced from Sigma-Aldrich. For pH adjustment, hydrochloric acid (HCl, 37%) from VWR and sodium hydroxide (NaOH, anhydrous pellets) from Carlo Erba Reagents were used.

After 24 hours of spheroid formation, the spheroids were transferred into new conventional culture dishes to allow the spheroids to attach and spread on the bottom of the culture dish to grow as a monolayer. When CBDCs in monolayer culture or spheroids reached 50-60% confluence, the basic culture medium was replaced with osteogenic induction medium (basic culture medium, supplemented with 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 50 μM L-ascorbic acid phosphate (Wako Pure Chemical Industries, Ltd.), and 10 mM glycerol phosphate disodium salt hydrate (Sigma-Aldrich)) or neurogenic induction medium (basic culture medium, supplemented with 50 ng/ml recombinant nerve growth factor, 50 ng/ml recombinant brain-derived neurotrophic factor, and 10 ng/ml recombinant NT-3 (all three reagents from PeproTech, Rock Hill, NJ, USA)). During the induction process, the media were changed every two days.