Glycerol phosphate

When the cells were 40–50% confluent, the culture medium was replaced with α-MEM containing dexamethasone (10 nmol·L⁻¹, Sigma-Aldrich Co., St. Louis, MO, USA), glycerol phosphate (5 mmol·L⁻¹, Sigma-Aldrich), penicillin (100 units per mL, Gibco-BRL), streptomycin (100 μg·mL⁻¹, Gibco-BRL), amphotericin B (0.25 μg·mL⁻¹, Gibco-BRL), l-ascorbic acid (100 μmol·L⁻¹, BioBasic Inc.) and 10% FBS (Equitech-Bio Inc.) for osteogenic differentiation of the cells in the presence or absence of 100 ng·mL⁻¹ LPS and TGF-β2 inhibitor (R&D Systems, Minneapolis, MN, USA). On day 7, ALP activity was measured using a QuantiChrom assay kit (BioAssay Systems, Hayward, CA, USA) according to the instructions provided by the manufacturer. 14–28 days after induction, the cells were stained with 40 mmol·L⁻¹ alizarin red S solution (pH 4.2, Sigma-Aldrich) for 15 min to visualise the calcium accumulation in mineralised cells. Quantification of alizarin red S was performed by destaining with a solution of 20% methanol and 10% acetic acid. The optical density of the solvent was read at an absorbance of 450 nm.

All the cells were cultured in vitro at 37 °C, 5% CO₂. MSC growth medium comprised of Dulbecco’s Modified Eagle’s Medium (DMEM) (Lonza, UK) supplemented with 10% (v/v) foetal bovine serum (FBS) (Sigma-Aldrich, UK), L-glutamine final media concentration 2 mM (ThermoFisher Scientific, UK), and 100 units/ml penicillin-streptomycin (ThermoFisher Scientific, UK). MSC osteogenic medium comprised of MSC growth media supplemented with 100 nM dexamethasone (Sigma Aldrich, UK), 10 mM glycerolphosphate (Sigma Aldrich, UK) and 50 μg/ml L-ascorbic acid (Sigma Aldrich, UK). RAW growth medium comprised of α-MEM (Life Technologies, NZ) supplemented with 10% (v/v) FBS (Life Technologies, NZ), L-glutamine final media concentration 2 mM (Life Technologies, NZ) and 100 units/ml penicillin-streptomycin (Life Technologies, NZ). RAW cell differentiation medium comprised of growth media supplemented with 10 ng/ml RANK-L (Amgen). Mature osteoclast (MO) growth medium comprised of Earle’s MEM (ThermoFisher Scientific, NZ) supplemented with 10% (v/v) FBS, 100 units/ml penicillin-streptomycin and 0.1% 12 M HCL.

Eight horses were used for differentiation experiments. For tenogenic control cultures, the cells were cultivated in expansion medium with a seeding density of 10,000 cells/cm². The medium was renewed every 2–3 days and the cells were kept in a 5% CO₂ environment at 37°C. To induce differentiation, we followed protocols established by others [22] . Briefly, to induce osteogenic differentiation, the cells were seeded at a density of 3,000 cells/cm² and cultured in DMEM High Glucose with Glutamax (Life technologies), 10% FCS (Life technologies), 0.6% fungizone (Life technologies), 0.1% gentamicin (Life technologies) and freshly added 10 mM glycerol phosphate (Sigma-Aldrich), 0.1 μM dexamethasone (Sigma-Aldrich) and 0.1 mM L-ascorbic acid 2-phosphate (Sigma-Aldrich). To induce adipogenic differentiation cells were seeded at 20,000 cells/cm² and the induction medium consisted of DMEM Glutamax (Life technologies) with 10% FCS (Life technologies), 0.6% fungizone (Life technologies), 0.1% gentamicin (Life technologies) and freshly added 0.1 μM dexamethasone (Sigma-Aldrich), 0.2 mM indomethacin (Sigma-Aldrich), 0.01 mg/ml insulin (Sigma-Aldrich) and 0.5 mM 3 iso-butyl-1-methyl-xanthine (Sigma-Aldrich). The cells were kept for 21 days in a 5% CO₂ environment at 37°C and the differentiation media were refreshed twice a week.

To induce osteogenic differentiation, cells of the 5th to the 19th passage were treated with osteogenic medium for 12 days. The differentiation medium was changed every 2 days. Osteogenesis was assessed at weekly intervals. The osteogenic medium consisted of IMDM supplemented with 0.01 μM dexamethasone (Sigma-Aldrich, St Louis, MO, USA), 50μM glycerol phosphate (Sigma-Aldrich, St. Louis, MO, USA), and 0.2 mM ascorbic-2-phosphate (As2P; Sigma-Aldrich, St. Louis, MO, USA) [20 (link),35 (link),36 (link),37 (link),38 (link),39 (link),40 (link),41 (link),42 (link),43 (link),44 (link),45 (link),46 (link),47 (link),48 (link),49 (link),50 (link),51 (link),52 (link),53 (link),54 (link),55 (link),56 (link)]. The osteogenic differentiation of HBMSs was detected by Alizarin red staining. Briefly, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After washing once with ddH₂O, 0.5 mL alizarin red S solution (2%, pH 4.2) was added to each well in a 24-well plate. The staining solution was removed after 10 min. Each well was washed with ddH₂O twice. The plate was then air-dried at room temperature and mineralization was detected by microscopy.

For osteogenic differentiation, cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS supplemented with 0.2 mM ascorbic acid, 10^–7 M dexamethasone, and 10 mM glycerol phosphate (Sigma-Aldrich, MO, USA) for 3 weeks, and the mineralization capacity was evaluated by von Kossa staining. Calcium deposition was quantified according to the instructions of the calcium colorimetric assay kit (BioVision, San Francisco, CA, USA). For adipogenic differentiation, the cultures were incubated in DMEM supplemented with 10% FBS, 10^–6 M dexamethasone, 0.5 μM isobutyl-methylxanthine, 10 ng/mL insulin, and 60 μM indomethacin (Sigma-Aldrich, MO, USA) for 2 weeks. Cells were fixed with paraformaldehyde and stained with fresh Oil Red O solution (Sigma-Aldrich, MO, USA).

Osteogenic differentiation was induced in 3D scaffold cultures in the presence of osteogenic medium, a-MEM supplemented with 15% FBS, 2 mM glutamine, 0.1 mM L-ascorbic acid phosphate, 100 U/mL penicillin, 100 mg/mL streptomycin, 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 1.8 mM KH₂PO₄, and 5 mM glycerolphosphate (Sigma-Aldrich, St. Louis, MO, USA). After 7 days culture in MSC expansion medium the cell-seeded scaffolds were induced to differentiate into osteocytes by using osteogenic medium for 21 days (the medium was changed twice per week). The morphology of the UG scaffolds seeded with hADMSCs after 21 days culture in osteogenic medium was observed with SEM. Scaffolds were fixed with 3% v/v glutaraldehyde, rinsed, and then dehydrated in increasing concentrations (30–100% v/v) of EtOH in water. The samples were dried and observed without sputter coating.