Ascorbic acid 2 phospate

A total of 1.5 × 10⁴ HUCMSCs were seeded in 6-well-plate for the above treatments. After 7 days, the HUCMSCs were seeded in a 15 ml conical tube at a density of 1 × 10⁶ cells cm⁻² and grown in chondrogenic media consisting DMEM, 10% FBS, 10 ng ml⁻¹ transforming growth factor-β1 (Pepro Tech, Rocky Hill, NJ, USA), 50 μg ml⁻¹ ascorbic acid-2-phospate (Sigma-Aldrich) and 6.25 μg ml⁻¹ of insulin (Sigma-Aldrich). The media was changed every 3 days. The cells were incubated with the chondrogenic media at 37 °C with 5% CO₂ for 3 weeks. After fixing in paraformaldehyde (Bionovas, Toronto, ON, Canada), the pellets were sliced into 5-μm sections, mounted on slides and stained by immunohistochemistry using type 2 collagen (Sigma-Aldrich).

MC3T3-E1 Subclone 4 osteoblast cell lines were acquired from ATCC (ATCC CRL-2593). Cells were cultured in αMEM + Glutamax (32571-036; Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% Pen/Strep (Corning). To stimulate procollagen synthesis and secretion, ascorbic acid 2-phospate (Sigma-Aldrich) was supplemented 18–24 h before imaging experiments.
Primary osteoblasts were extracted from mice harboring the G610C mutation and their wild-type littermates (B6.129(FVB)-Col1a2tm1Mcbr/J; Jackson Laboratories), which were maintained on the C57BL/6J background (4 (link), 23 (link)). Osteoblasts were extracted from parietal bones of 3- to 8-d-old mice as previously described (4 (link)). All care and procedures were performed in accordance with a Eunice Kennedy Shriver National Institute of Child Health and Human Development Animal Care and Use Committee-approved protocol.