Fig 1: (A–D) Von Kossa stained decellularized resorption plates of MC-MSC co-cultures. (E) Quantification of resorbed area, p<0.05 (Kruskal-Wallis and Dunn’s post hoc tests). (F) TRAP activity quantification, p < 0.05 for culture time, culture condition, no interaction effect (two-way ANOVA and Turkey’s post hoc tests within each time point). (G) Micrographs of MC-MSC co-cultures stimulated to undergo osteoclastic differentiation, stained for F-Actin (red), the nucleus (gray), OPG (blue) and RANKL (green). Images were taken at locations rich of MSCs to detect their influence on OPG and RANKL production. Scale bar in insert is 20 µm. (H) Quantification of OPG in supernatant, dashed lines represents concentration in the medium control, p < 0.05 (one-way ANOVA and Turkey’s post hoc tests). (I) Quantification of RANKL in supernatant, dashed line represents concentration exogenous RANKL added to the medium, p < 0.05 (one-way ANOVA and Turkey’s post hoc tests). (J) RANKL/OPG ratio in supernatant, p < 0.05 (Kruskal-Wallis and Dunn’s post hoc tests). Asterisks in figures represent results of post hoc analyses (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). fetal bovine serum (FBS), human platelet lysate (hPL), monocytes (MCs), mesenchymal stromal cells (MSCs), tartrate resistant acid phosphatase (TRAP), osteoprotegerin (OPG), receptor activator of nuclear factor κB ligand (RANKL).
Fig 2: Adding native stromal cells promotes ameloblastoma invasion compared to acellular. Invasion of AM-1 cells from the tumour mass to (A) acellular (A), (B) MRC5, (C) and to (D) hOB stroma at day 14, red = Phalloidin, green = RANKL, blue = DAPI, scale bar = 100 µm. White lines describe tumour mass and stroma boundary and yellow lines represent invasion of tumour cells to the surrounding stroma. (E&F) Invasion distance of AM-1 and AM-3 cells to HFG and MRC5 stroma at day 14. mRNA expression of (G&H) TNFSF11 (RANKL) in AM-1 and AM-3 tumoroids with acellular stroma, MRC5, HGF, and hOB stroma and controls of HGF and hOB only tumouroids at day 14. (I) RANKL protein concentration in AM-1 and AM-3 tumouroids at day 14. All values were measured as n = 3. One-Way ANOVA, Dunnet’s Post Hoc; p-values 0.05 < *, 0.005 < **, 0.0005 < *** and 0.00005 < ****. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 3: RANKL expression of the ameloblastoma 3D tumouroid models, where (A) AM-1 tumouroid with acellular stroma (total magnification 400×), (B) AM-1 tumoroid with HGF stroma (total magnification 200×), (C) AM-3 tumouroid with acellular stroma (total magnification 400×), and (D) AM-3 tumoroid with HGF stroma (total magnification 200×). RANKL expression quantification for the (E) tumour and (F) stromal region by using the QuPath software. A significant expression can be observed between the ameloblastoma cell lines (AM-1 vs AM-3) and type of stroma (acellular vs HGF stroma), including stromal expression. Data presented with mean ± SEM, p-values 0.05 < *, 0.005 < **, 0.0005 < ***. N = 3 with 5 random regions of interest were selected.
Fig 4: Depletion of NRP2 escalates the resorptive function and gene expression in osteoclasts. Knockout of NRP2 in osteoclastic precursors isolated from NRP2 Fl/Fl; CSF1R-Cre transgenic mice by addition of 4-HydroxyTamoxifen in vitro and differentiated into osteoclasts in the presence of RANKL and M-CSF, LNCaP C4-2B CM and PC3 CM on a 24-well osteoassay plate. a–c Representative images of pit resorption to compare the resorbed area in NRP2WT and NRP2KO osteoclasts in RANKL+M-CSF, LNCAP C4-2B CM and PC3 CM respectively. d–f Quantification and comparison of the percentage resorbed area in NRP2WT and NRP2KO osteoclasts in RANKL and M-CSF, LNCaP C4-2B CM and PC3 CM respectively. g–i Graphical representation of the osteoclastic gene expression at mRNA in NRP2WT and NRP2KO condition treated respectively with RANKL and M-CSF, LNCaP C4-2B CM and PC3 CM. All values reported as mean± SEM from three independent experiments and represented as a bar graph with error bar. Statistically significant P value denoted as * (0.05), ** (0.01), *** (0.001)
Fig 5: NRP2 ablation increases osteoclast differentiation. a Protein analysis of NRP2 expression in a time course (0-6 days) in osteoclasts differentiated under different conditions in comparison to osteoclastic precursors under the conditions of RANKL (100 ng·mL−1) and M-CSF (20 ng·mL−1), LNCaP C4-2B CM and PC3 CM. Right: Comparison of NRP2 protein levels in RANKL and M-CSF, LNCaP C4-2B CM and PC3 CM at day 6. b Graph showing the expression of NRP2 at mRNA level in all the three conditions in the time course of osteoclast differentiation. Knockout of NRP2 in osteoclastic precursors isolated from NRP2 Fl/Fl; CSF1R-Cre transgenic mice by addition of 4-HydroxyTamoxifen in vitro and differentiated into osteoclasts in the presence of RANKL and M-CSF, LNCaP C4-2B CM and PC3 CM. c–e TRAP staining showing osteoclast differentiation at day 4–6 after depletion of NRP2 in RANKL and M-CSF, LNCaP C4-2B CM and PC3 CM, respectively. f–h Quantification of the TRAP positive multinucleated cells per well and comparison between NRP2WT and NRP2KO osteoclasts represented as a graph in RANKL and M-CSF, LNCaP C4-2B CM and PC3 CM, respectively. All values reported as mean± SEM from three independent experiments. Statistically significant P value denoted as * (0.05), ** (0.01), *** (0.001)
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