Fig 1: BP effect on protein secretion by pooled human primary pre-osteoblasts. OPN (A) SOST(B) DKK-1 (C) TNFa (D) IL-1ß (E) RANKL and OPG (F) release was evaluated at both day 3 and 7 after ALN or ZOL treatment. Data are represented as relative values setting as 100% the secretion levels of the different analytes by untreated pre-osteoblasts (ctrl, blue dashed lines). Values are expressed as mean ± SEM of 4 (pool 1-4) and 3 (pool 2-4) independent experiments at each time point (n=12 and n=9 cell populations respectively). For each pool, donor characteristics are described in Table 1. Statistical significance versus ctrl is shown as *p<0.05.
Fig 2: Introduction of ameloblastoma tumour mass to the 3D bone stroma prior to day 9 completely inhibits bone nodule formation. (a) Introduction of AM-1 and AM-3 tumour masses to 3D bone at day 6 inhibits bone nodule formation. Images taken at day 6, 9 and 21, 4 × Magnification, scale bar = 100 µm. ALPL expression. TNFSF11 (RANKL) Expression. (b) Introduction of AM-1 and AM-3 tumour masses to 3D bone at day 9 restricts bone formation by limiting bone nodule number and bone nodule surface area. (c) RT2 Profiler PCR Array was conducted to screen osteogenesis gene of osteoblasts in the 3D bone stroma model and in AM-3 tumour mass introduced 3D bone stroma model at day 8. The AM-3 tumour mass was introduced at day 6 of 3D bone stroma model. Volcano plot shows under-expressed, unchanged and over-expressed genes. The table represents > 3.5-fold under-expressed gene. Horizontal line p-value threshold (0.05). One-Way ANOVA, Dunnet’s Post Hoc; p-values 0.05 = *, 0.005 = **, 0.0005 = *** and 0.00005 = ****.
Fig 3: Characterisation of ameloblastoma cells in 3D culture. Spheroid formation of (a) AM-1 cells and (b) AM-3 cells in 3D tumouroids at day 7, red = Phalloidin, blue = DAPI, 20 × Magnification Scale Bar = 50 µm. (c) Histology H&E staining of plexiform patient samples, (d) 3D AM-1 tumouroids, e follicular patient samples, and (f) 3D AM-3 tumouroids. Similar anastomosing cords and branches were highlighted (c, d) 10 × Magnification Scale Bar = 100 µm. The formation of the odontogenic islands was highlighted (e,f). (g) CellTiter-Glo 3D Viability-Assay of AM-1, AM-3 and MG-63 cells in 3D tumouroids. Distance of invasion (µm) AM-1, AM-3 and MG-63 cells from the tumour mass to the surrounding stroma within the 3D tumouroids. (h) Human MMP-2 ELISA. Expression of MMP2. (i) Human MMP-9 ELISA. Expression of MMP9. (j) Expression of TNFRSF11A (RANK). Human RANKL ELISA. One-Way ANOVA, Dunnet’s Post Hoc; p-values 0.05 = *, 0.005 = **, 0.0005 = *** and 0.00005 = ****.
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