Complete egm 2

Mouse C₂C₁₂ myoblast cells and mouse endothelial cells 2H11 were maintained in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), and penicillin/ streptomycin (Invitrogen). For 2H11 cells, the plates were pretreated with 0.1% gelatin (Sigma‐Aldrich, St Louis, MO, USA). Mouse osteoprogenitor cells KS483 were cultured in α‐MEM (Gibco) and 10% FBS (Invitrogen), and penicillin–streptomycin (Invitrogen). Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured in complete EGM‐2 (Lonza, Walkerville, MD, USA) medium supplemented with 10% FBS and 0,1% penicillin–streptomycin. Mouse chondrogenic ATDC5 cells were cultured in phenol red free DMEM‐F12 (Gibco) containing 5% FBS. All of the cells were grown at 37°C in a humidified incubator with 5% CO₂. BMP‐6 was a kind gift from Dr S Vukicevic. TGF‐β3 was provided by Dr A Hinck. Activin A was purchased from R&D Systems (Abingdon, UK). TNF‐α was purchased from Pierce (Thermo Fisher Scientific, Waltham, MA, USA).

HUVECs were seeded (5000 cells per well) in a 96‐well plate into 200 μL of complete EGM‐2 (Lonza, Walkersville, MD, USA). Post 24 h of seeding, old media was replaced with a cocktail of EGM‐2 and conditioned media (1 : 1 ratio) [15 (link)]. After 24 h, the cocktail was replaced with 100 μL of fresh EGM‐2 media and 10 μL of CellTiter 96® AQueous One Solution Reagent (Promega, Madison, WI, USA) and incubated for an hour inside the incubator at 37 °C and 5% CO₂. Afterward, absorbance was recorded in Varioscan multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 490 nm.