Rhbmp6

For cell stimulation with rhBMP6 (S. Vukicevic, University of Zagreb, Croatia), rhBMP9 (PeproTech, Hamburg, Germany), rhTGFβ-1 (PeproTech), and rhActivin-A (R&D Systems), growth factors were reconstituted and stored according to manufacturer instructions. For small-molecule–based inhibition (SMKI), K02288 (Alex Bullock, Oxford, UK) [120 (link)] and SB-431542 (Sigma-Aldrich) [47 (link)] were added to cells 1 h prior to ligand stimulation with indicated concentrations unless stated otherwise. For long-term Rho-kinase inhibition, Y-27632 (Stemcell Technologies) was added at the indicated concentration at time of cell-seeding and 12–48 h later.

For chondrogenic differentiation, chondrocytes were cultivated for 28 days in CDM (DMEM 4.5 g/L glucose and Ham’s F12 (1:1), 100 U/mL penicillin/streptomycin, 40 ng/mL L-proline, 2 mM L-glutamine, 1 mM Na-pyruvate, 0.1 µM dexamethasone, 50 μg/mL ascorbic acid, 10 μL/mL ITS [Sigma-Aldrich], 10 ng/mL rhTGF-β3 [Peprotech], 10 ng/mL rhBMP6 [Peprotech]) under serum-reduced (in case of 2D culture; 1% FSC) or serum-free (in case of pellet culture) conditions as previously described (Riegger et al., 2018b (link)). For osteogenic differentiation, chondrocytes were cultured for 28 days in ODM (DMEM 1 g/L glucose, 10% FBS, 100 U/mL penicillin/streptomycin, 2 mM L-glutamine, 0.1 µM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate).

Sorted KCs by FACS were seeded in 12-well plated in DMEM (HyClone) containing 10% FBS (Gibco, Thermo Fisher Scientific). After overnight, the culture medium was replaced by serum-free X-VIVO15 media (Lonza), supplemented with 20 ng/mL M-CSF in the presence or absence of 50 ng/mL rmBMP2 (Peprotech), rhBMP6 (Peprotech), rhBMP9 (Peprotech), or 50 ng/mL rmBMP10 (R&D Systems). The half media were changed every other day. On day 7, KCs were acquired, and RNA was extracted using an RNeasy Plus Mini Kit (QIAGEN).