Lentiviral production was performed in HEK293T cells using the Lenti-vpak Lentiviral Packaging Kit (Origene Technologies, Rockville, MD 20850, USA). In short, HEK 293 T cells were expanded in DMEM high glucose (Life Technologies Europe BV, Bleiswijk, The Netherlands), supplemented with 10% foetal calf serum (FCS; Life Technologies Europe BV, Bleiswijk, The Netherlands) 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies Europe BV, Bleiswijk, The Netherlands), and lentivirus particles were collected and titrated.
Following their isolation, hPACs were transduced at passage 1 with either control (pLV.CMV.bc.eGFP) or TNFRSF11B Lentivirus (MOI of 1). After 16 h, the medium was refreshed [DMEM high glucose (Life Technologies Europe BV, Bleiswijk, The Netherlands) supplemented with 10% FCS (Life Technologies Europe BV, Bleiswijk, The Netherlands), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies Europe BV, Bleiswijk, The Netherlands), and 0.5 ng/ml bFGF-2 (PeproTech, London, UK)] and hPACs were further cultured for another passage. Subsequently, neo-cartilage was generated from 250 000 transduced hPACs in 3 D pellets for seven days, as described before [12 (link)], and keeping the conditions between both groups equal. All data were analysed 7 days following the 3D chondrogenesis.
Following their isolation, hPACs were transduced at passage 1 with either control (pLV.CMV.bc.eGFP) or TNFRSF11B Lentivirus (MOI of 1). After 16 h, the medium was refreshed [DMEM high glucose (Life Technologies Europe BV, Bleiswijk, The Netherlands) supplemented with 10% FCS (Life Technologies Europe BV, Bleiswijk, The Netherlands), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies Europe BV, Bleiswijk, The Netherlands), and 0.5 ng/ml bFGF-2 (PeproTech, London, UK)] and hPACs were further cultured for another passage. Subsequently, neo-cartilage was generated from 250 000 transduced hPACs in 3 D pellets for seven days, as described before [12 (link)], and keeping the conditions between both groups equal. All data were analysed 7 days following the 3D chondrogenesis.
