β glycerophosphate

Osteogenic differentiation was performed according to^{104 (link)} with minor modifications. Briefly, WT and mutant MenSCs at passages 4–7 were plated at a density of 10,000 cells/ cm² in 12-well plates in a regular culture medium. After 72 h, the culture medium was replaced by osteogenic differentiation medium containing high-glucose DMEM (Sigma), 10% FBS, 1 µM dexamethasone (Alfa Aesar, cat # A17590), 250 µM sodium ascorbate (Sigma, cat # A4034), and 10 mM β-glycerophosphate (Alfa Aesar, cat # L03425). The medium was changed every 3–4 days. Control cells were kept in regular culture medium (RCm). After 20 days of induction, cells were fixed in 4% FA and incubated with the Mouse Anti-human osteocalcin monoclonal antibody (1:500; R&D, cat# MAB1419) followed by incubation with anti-mouse DyLight™ 594 secondary antibody (1:500) and 1 μM Hoechst 33,342 (Life Technologies).

The MC3T3-E1 subclone 4 pre-osteoblast, mouse (Mus musculus) -derived cell line was purchased from ATCC-CRL-2593. Minimum Essential Eagle’s Medium (α-MEM) (Corning, Corning, NY, USA), ascorbic acid (Avantor, Gliwice, Poland), β-glycerophosphate (Cayman Chemical, Ann Arbor, MI, USA) 100 units penicillin, 100 µg streptomycin, 250 ng amphotericin B per mL (ABAM, Sigma, St. Louis, MO, USA), fetal bovine serum (FBS, Biowest, Nuaillé, France), and trypsin/EDTA (Biowest) were also obtained [23 (link)]. We also used proliferation medium: α-MEM, 10% FBS, 1%vol. ABAM; differentiation medium: α-MEM, 10% FBS, 1%vol. ABAM, β-glycerophosphate 10 mM, ascorbic acid 50 µg/mL. Diclofenac (DF, J62609, Alfa Aesar, Kandel Germany, diclofenac sodium salt CAS number 15307-79-650) 50 mM stock in DMSO (dimethyl sulfoxide, sterile, catalogue number, DMS666, CAS Number 67-68-5 BioShop Canada Inc., Mainway Burlington, Ontario, Canada), final concentrations in differentiation culture media 50, 5, and 0.5 µM, methylprednisolone (MP, PHR1717-500MG, Merck, Darmstadt, Germany, C.A.S. Number 83-43-2) 50 mM stock in DMSO, final concentrations in differentiation culture media 50, 5 and 0.5 µM were also used.

Osteogenic differentiation was performed according to [35 (link)] with minor modifications. Briefly, WT and mutant cells at passages 4–7 were plated at a density of 10,000 cells/ cm² in 12-well plates in regular culture medium. After 72 h, the culture medium was replaced by osteogenic differentiation medium containing high-glucose DMEM (Sigma), 10% FBS, 1 μM dexamethasone (Alfa Aesar, cat # A17590), 250 μM sodium ascorbate (Sigma, cat # A4034), and 10 mM β-glycerophosphate (Alfa Aesar, cat # L03425). The medium was changed every 3–4 days. Control cells were kept in regular culture medium. After 28 days of induction, cells were fixed in 4% FA and stained with standard Von Kossa Staining.

For osteogenic differentiation, the cells were seeded on the scaffolds (1 × 10⁵ cell/well in 24-well plate), and, after 2 days, the culture medium was replaced by an osteogenic differentiation medium. The composition of the differentiation medium was as follows: StemPro^® Osteocyte/Chondrocyte Differentiation Basal Medium, StemPro^® Osteogenesis Supplement, 1% gentamicin solution, 10 nM dexamethasone, 10 mM β-glycerophosphate, 50 μM ascorbate-2-phosphate (GIBCO™ Invitrogen Corporation, Carlsbad, CA, USA). The medium was replaced with a fresh portion every 3 days for 14 days.

Human dental pulp (HDP) fibroblasts and MC3T3 osteoblastic cells (Kargarpour et al. [21 (link)]) were seeded onto disks with 6 mm in diameter and 1 mm thick of each material in 96-well plates at a density of 10,000 cells per well either group. During cell seeding, α-MEM medium (Invitrogen, Basel, Switzerland) was supplemented with 50 μg/mL ascorbic acid (Invitrogen) and 2 mM β-glycerophosphate (Invitrogen) to promote proliferation/differentiation [4 (link)]. Cells were quantified using fluorescent MTT assay (Invitrogen) at 1, 3, and 5 days for cell proliferation as previously described ²¹. At desired time points, cells were washed with phosphate-buffered solution (PBS) and quantified using a fluorescence plate reader (Infinite 200, Tecan, Männedorf, Switzerland). Experiments were performed in triplicate with three independent experiments for each condition. Qualitative analysis of resulting cells was obtained by LIVE/DEAD dyes (Sigma Aldrich) using confocal-laser scanning microscopy [4 (link)] .

ST2 mesenchymal stromal cells isolated from mouse bone marrow were obtained from RIKEN Cell Bank (Tsukuba, Japan), and were grown in DMEM medium supplemented with 10% fetal calf serum (FCS; Invitrogen, Zug, Switzerland). For the differentiation experiments, media were supplemented with 50  ascorbic acid (Invitrogen) and 2 mM β-glycerophosphate (Invitrogen), as described.^{9 (link)} Cells were treated with either fivefold-diluted BCM or recombinant TGF-β1 and/or BMP-2 proteins (PeproTech, London, UK) for 30 min (protein analyses), 24 h (RNA analyses), or 4 days (mineralization analyses).
Proliferation rates of BCM-treated ST2 cells were determined using a BrdU incorporation assay (Roche, Basel, Switzerland), as described.^{37 (link)} In brief, after 24 h of starvation, the cells were plated in triplicate on black 96-well plates (PerkinElmer, Basel, Switzerland) at 2 × 10³ cells/well in 3% FCS/DMEM and were allowed to proliferate for 0, 24, 48, 72, and 96 h before labeling with BrdU for 2 h. BrdU incorporation into newly synthesized DNA was determined according to the manufacturer’s protocol using an Infinite® 200 luminometer (Tecan, Männedorf, Switzerland). Experimental values were normalized to the values of ST2 cells treated with RS or RS+S at time point 0. Data represent means±SD from four independent experiments.