W 54011

MSCs were seeded overnight at (3000 cells/cm 2 ) in 96-well microplates. Cells were incubated with conditioned media for 5 min. In parallel, cells were also pre-incubated for 5 min with or without 10 nM of W54011, a specific C5aR antagonist (Merck, Darmstadt, Germany). After washing, cells were fixed (3% paraformaldehyde, 20 min) and saturated (5% BSA, 1 h). C5a/C5aR binding was visualized by biotinylated mouse anti-human C5a Supernatants from injured PDL cells incubated with materials' extracts (conditioned media) were applied on MSCs cultures to evaluate C5a binding to MSCs C5aR and its phosphorylation by ELISA, to quantify their proliferation using MTT assay and their migration using Boyden chambers IgG (2 μg/mL, 2 h) followed by Streptavidin-HRP (20 min) and substrate solution (20 min) (R&D Systems, Lille, France). C5aR phosphorylation was visualized by biotinylated rabbit antihuman C5aR-p(Ser338) (Abcam, Paris France) (2 μg/mL, 2 h) followed by Streptavidin-HRP (20 min) and substrate solution (20 min) (R&D Systems, Lille, France). Optical densities were measured at 650 nm using an automatic microplate spectrophotometer (Metertech Inc., Taipei, Taiwan). C5a/C5aR binding and C5aR phosphorylation are expressed as percentage of the control condition.