β glycerophosphate disodium salt hydrate

Graphene oxide (GO) was obtained from Graphenea (Donostia, Gipuzkoa, Spain). Doxorubicin hydrochloride salt (DOX, molecular weight: 579.98 Da) was purchased from LC laboratories (Woburn, MA, USA). Chitosan (CS, medium molecular weight 190–310 kDa, deacetylation ≥ 75%), β-glycerophosphate disodium salt hydrate (GP, molecular weight = 216.04 Da), cellulose dialysis membrane (molecular weight cut-off (MWCO) 14 kD), acetic acid, absolute ethanol, Eagle’s minimum essential medium (MEM), Dulbecco’s Modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin/ ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS) and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO, USA).

The multipotent MSC D1 cell line was differentiated into adipocytes and osteocytes. For that purpose, the medium was exchanged immediately after labelling and replaced with fresh medium containing adipogenic or osteogenic supplementation. Adipogenic supplementation consisted of 100 nM Dexamethasone, 10 mM β-glycerophosphate disodium salt hydrate and 77 µM 2-Phospho-L-ascorbic acid trisodium salt (all from Sigma), whereas the osteogenic supplementation consisted of 100 nM Dexamethasone, 155 µM 2-Phospho-L-ascorbic acid trisodium salt, 50 µM indomethacin and 175 nM bovine pancreas insulin (all from Sigma). The cells were then allowed to grow for a further 9 d with periodic medium changes. Effective differentiation was evaluated by fixation and histochemical staining with 0.5% Oil Red O (Sigma) in isopropanol or 2% Alizarin Red S (Sigma).

To assess the calcium deposition by osteoblasts, BMSCs were cultured in osteogenic differentiation‐inducing medium for 14 days. After osteogenic differentiation, the cells were stained with alkaline phosphatase (ALP) staining solution for 4 hours at 37°C using published protocol. The ALP staining solution was a mixture of 3% β‐glycerophosphate disodium salt hydrate (Sigma, USA), 2% sodium pentobarbital (Peking Tongxian Yucai fine chemical, China), 2% calcium chloride (Tianjin Bodi Chemical, China) and 2% magnesium sulphate (Tianjin Haijing Fine Chemical, China). The cells were incubated with ALP staining solution for 4 hours at 37°C. After incubation four 4 hours, 2% cobalt nitrate was quickly added to the plates for 5 minutes and the cells were rinsed three times with PBS. Then the cells were incubated in 0.2% ammonium chloride solution for 1 minute. Thereafter, 10 pictures were randomly taken and calcium‐rich deposits were observed under a microscope (Nikon, Japan). The gradation of colour represented the intensity of calcification of cells.

Assembling of cells and particles to microtissues followed a previously described protocol [34 (link)]. Briefly, hMSC (Lonza, Basel, Switzerland) were cultivated in DMEM low glucose (Sigma Aldrich, Seelze, Germany), 10% (v/v) fetal bovine serum albumin (FBS, Biochrome, Berlin, Germany), 1% amino acids and antibiotics and detached the cells with Trypsin/EDTA (Sigma Aldrich, Seelze, Germany). We added 100 µL of cell suspension (10⁵ cells ml⁻¹) to 100 µL of cGM suspension (0.64 mg ml⁻¹) in Opti-MEM™ with 10% FBS per well of a low adhesion 96-well plate (Spheroid microplate, Corning, NY, USA). After 24 h, the medium was changed to osteogenic medium (OM) based on DMEM, 10% (v/v) FBS, 100 ng/mL Dexamethasone (Sigma Aldrich, Seelze, Germany), 50 µg/mL ascorbic acid (Sigma Aldrich, Seelze, Germany), 10 mM β-Glycerophosphate disodium salt hydrate (Sigma Aldrich, Seelze, Germany) with (OM^+BMP-2)/without (OM) 100 ng/mL bone morphogenetic protein (BMP-2, R&D Systems, Wiesbaden, Germany). Medium was changed partially by exchanging 200 µL of culture medium twice for three times a week in order to keep cGM/hMSC and microtissues in the well.

Undifferentiated iPSCs were cultured on iMEFs feeder in iMEF-conditioned hPSC medium described above. Cells were incubated with Collagenase IV (Sigma Aldrich) for 5 minute at 37 °C. Cells were then washed with culture medium 3 times, and the colonies were broken using a cell scraper (Corning). Cells were grown in ultralow attachment plates (Corning) as aggregates in mesodermal induction medium consisting of IMDM (Gibco) supplemented with 20% FBS, 0.1 mM NEAA, 2 mM L-glutamine, 50 U ml⁻¹ penicillin and 50 mg ml⁻¹ streptomycin, 4.5 mM 1-Thioglycerol (Sigma Aldrich), 50 μg/ml L-ascorbic acid-2-phosphate and 200 μg/mL holo-Transferrin human (Sigma Aldrich). Medium was carefully refreshed every 2 days. At day 5, aggregates were replated in Gelatine (Sigma Aldrich) coated plates in osteogenic induction medium, consisting of Mem Alpha (Gibco) supplemented with 10% FBS, 100 nM Dexamethasone, 10 mM β-Glycerophosphate disodium salt hydrate (Sigma), 50 μM L-ascorbic acid-2-phosphate, 2 mM L-glutamine and 50 U ml⁻¹ penicillin and 50 mg ml⁻¹ streptomycin. Medium was changed every 2 days. At day 21 of osteogenic differentiation, experiments were stopped and molecular characterizations carried out.