β glycerophosphate

The cell culture flasks and plates were coated with 0.1% gelatin from bovine skin (Sigma-Aldrich, St. Louis, MO, USA) prior to osteogenic differentiation in order to allow better JPC adherence and avoid detachment from the culture flasks during long-term incubation. For osteogenic differentiation, JPCs were cultured in osteogenic medium (DMEM/F-12+GlutaMAX medium, 10% hPL, 1% Pen-Strep, 1% amphotericin B, 100 µM L ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β glycerophosphate (AppliChem, Darmstadt, Germany) supplemented with or without 4 µM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) for 10 days for secretome collection or for 15 days for the analysis of the JPC osteogenic potential.

Uncoated or PLGA-coated β-TCP tricalcium phosphate scaffolds were placed in each well of a 96-well plate and preconditioned with 200 μL hPL5-medium at 37 °C. After 1 h, the medium was aspirated and each scaffold was seeded with 5 × 10⁴ JPCs in 50 μL hPL5 medium. The cells were incubated for 2 h at 37 °C to allow cell adherence and then 150 μL medium was added. After 24 h, scaffolds seeded with cells were transferred into a new 96-well plate and 200 μL hPL10 medium (DMEM/F12 containing 10% hPL, 100 U/mL penicillin-streptomycin (Lonza, Basel, Switzerland), 2.5 μg/mL amphotericin B) or osteogenic induction medium (hPL10 containing 0.1 mM L-ascorbic acid 2-phosphate (Sigma-Aldrich, Taufkirchen, Germany), β-glycerophosphate (AppliChem, Darmstadt, Germany), and 4 μM dexamethasone (Sigma-Aldrich)) were added to each scaffold. Cells seeded onto β-TCP scaffolds were cultivated at 37 °C for 15 days with medium changes every 2–3 days.

Cells (30,000/foam) were seeded onto the sterilized unmodified, fibronectin modified, and laminin modified PBS foams. Cell seeded foams were incubated in a CO₂ incubator at 37°C, in 5% CO₂ and 90% humidity throughout 20 days. At day 3, osteogenic medium (Dulbecco's Modified Eagle Medium (DMEM: 4.5 g/liter glucose)) (Gibco Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco-Invitrogen, USA), 100 units/mL Penicillin-Streptomycin-Amphotericin (PSA) (Lonza, Switzerland), 50 μM ascorbic acid (AppliChem, Germany), 100 nM dexamethasone (AppliChem, Germany), and 10 mM β-glycerophosphate (AppliChem, Germany) was added into the wells and it was changed twice a week. All tests were carried out in triplicate throughout the whole study.