Bone morphogenetic protein signalling dynamics in hFOBs under two-dimensional and three-dimensional culture conditions. (a) hFOBs in two-dimensional monolayer cultures were stimulated with 5 nM BMP2 for indicated time points and protein phosphorylation was analysed by western blot using specific antibodies. (b) hFOBs in two-dimensional monolayer cultures were transfected with a BMP responsive reporter construct (BRE-Luc) and stimulated with different ligand concentrations for 24 hours. Bar charts depict means ± standard error of the mean of relative luciferase activity (RLA); n = 3; ***P < 0.001. (c) hFOBs were cultured on collagen scaffolds and cell morphology was assessed by immunofluorescent staining. Cell morphology was visualized by actin staining (red), cell nuclei were counterstained by DAPI (blue) and collagen matrix is depicted in green. (d) hFOBs were cultured on collagen scaffolds, stimulated with 10 nM BMP2 and protein phosphorylation was analysed by western blot using specific antibodies. BMP: bone morphogenetic protein; DAPI: 4'-6-diamidino-2-phenylindole; hFOB: human fetal osteoblast; RLA: relative luciferase activity.