L ascorbic acid 2 phosphate

A total of 50,000 osteoblasts were resuspended in 20 µL of DMEM/10%FBS/1%PS, seeded onto each PCL scaffold on the convex-shaped top side and incubated for 2 h at 37 °C, 5%CO₂. After 2 h, 1 mL of basal media was added to each well of the 24-well plate. The following day, to prevent attachment of the scaffold–cell constructs to the bottom of the tissue culture plate, each scaffold was transferred to a 15 mL Falcon tube (Corning) using sterile tweezers and underwent osteogenic induction with 2 mL of DMEM/10%FBS/1%PS supplemented with 50 µg/mL l-ascorbic acid-2-phosphate, 10 mM β-glycerophosphate and 0.1 µM dexamethasone (all Sigma Aldrich). Scaffolds were further cultured at 37 °C, 5%CO₂ in osteogenic media for up to 40 days.

Osteochondral explants were prepared as described for the fresh specimens, immediately placed in inserts, and washed with PBS until further use (Figure 1). The inserts are part of the culture platform used, which provided separate medium compartments for the bone and cartilage tissue (LifeTec Group BV, Eindhoven, the Netherlands).⁹ Explants were then cultured in separate wells provided with 3 mL bone medium, consisting of DMEM high glucose (14966; Gibco, Bleiswijk, the Netherlands) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin/streptomycin (Lonza, Basel, Switzerland), 25 μg/ml fungin (InvivoGen, Toulouse, France), 50 μg/mL l‐ascorbic acid‐2‐phosphate (Sigma‐Aldrich) and 10 mM β‐glycerophosphate (Sigma‐Aldrich) in the bone compartment and 2.5 mL cartilage medium, consisting of DMEM high glucose (14966) supplemented with 1% penicillin/streptomycin, 25 μg/mL fungin, 40 μg/mL l‐proline (Sigma‐Aldrich), 50 μg/mL l‐ascorbic acid‐2‐phosphate, and 1% ITS + Premix (Corning, Fisher Scientific, Landsmeer, NL) in the cartilage compartment. The medium was refreshed every 3 to 4 days and explants were cultured at 37°C and 5% CO_2.

HiPSCs were split at 1:8 to 1:12 ratios using EDTA, and grown for 3‐4 days until they reached ~85% confluence. The media were changed to CDM3,19 which consisted of RPMI 1640 (Corning), 500 µg/mL Oryza sativa‐derived recombinant human albumin (Oryzogen Sciencell), and 213 µg/mL L‐ascorbic acid 2‐phosphate (Sigma‐Aldrich). The media were changed every 48 hours. On day 0, the media were changed to CDM3 supplemented with 6 µmol/L CHIR99021 (MedChem Express).20, 21 On day 2, the media were changed to CDM3 supplemented with 2 µmol/L Wnt–C59 (Biorbyt). The media were changed on day 4 and every other day for CDM3. Contracting cells were observed from day 8. On day 10, the media were changed to a purification medium made using RPMI 1640 (no glucose) (Corning), 500 µg/mL recombinant human albumin, and 213 µg/mL L‐ascorbic acid 2‐phosphate. The medium was replaced with RPMI 1640 (Corning) supplemented with 500 µg/mL recombinant human albumin 48 hours before the experiment in order to avoid antioxidant effects.