Fig 2: JAG1 delivery in a PEG hydrogel stimulates bone regeneration in a critical-sized bone defect mouse model.HBO cells alone or in the presence of JAG1-Dynabeads complex (20 μM) ± DAPT and bone morphogenetic protein 2 (BMP2; 2.5 µM) + Fc-Dynabeads were incorporated in 4% PEG-MAL hydrogels and implanted into 4 mm critical-sized defects in the parietal bones of 6- to 8-week-old NOD SCID mice (n = 4–6 per HBO cell donor, 13–15 total) as two separate doses (Initial dose, week 4). After 8 weeks, we quantified differences in regenerated bone volume within the defect and compared them between experimental groups by micro computed tomography (μCT) analysis. (A) μCT reconstructions of defects. (B) Quantification of regenerated bone volume. Data are presented as mean (n = 13–15) ± standard deviation (SD) with p-values reported (one-way analysis of variance [ANOVA] with Šídák’s multiple comparisons test). (C) shows representative sections of the defect area on skulls from mice from all experimental groups stained with Masson trichrome stain.

Fig 3: JAGGED1 induces a non-canonical NOTCH pathway in HBO cells.HBO cells undergo mineralization through a non-canonical pathway. Luminex analysis of lysates obtained from three HBO cell lines untreated or treated with Dynabead-bound recombinant JAG1-Fc fragment (5.7 μM) ± DAPT (15 μM), a NOTCH canonical pathway inhibitor in a time course manner (5, 10, 15, and 30 min), (A) Heatmaps and (B) z-Scores plotted on graphs. Each data point represents mean n = 3 ± standard deviation (SD) per cell line with p-values reported.

Fig 4: JAGGED1-induced mineralization and gene expression in HBO cells: seven HBO cell lines were treated with growth media alone, osteogenic media alone or with Fc-Dynabeads (5.7 μM) or JAG1-Dynabeads (5.7 μM).The cells were half-fed every 5 days. On day 21 cells were fixed with 50% ethanol and thereafter, stained with 1% Alizarin Red S. (A) Representative image of HBO2. (B) Alizarin Red S dye was extracted from Alizarin Red S-stained cells using a 1:10 dilution of acetic acid and water, and the absorbance was read at 420 nm. Data represent the mean values of three technical replicates per cell line (mean ± standard deviation [SD], one-way analysis of variance [ANOVA] with Tukey post hoc). (C) HBO1 primary cell line was grown in triplicate and treated with growth media alone, osteogenic media alone or with Fc-Dynabeads (5.7 μM) or JAG1-Dynabeads (5.7 μM). The cells were half-fed every 5 days and collected at 7, 14, and 21 days. qRT-PCR was performed (see Methods). Data were normalized to growth media with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Data represent the mean values of three biological and two technical replicates per condition (mean ± SD, ordinary one-way ANOVA with Šídák’s multiple comparisons test, with single pooled variance).

Fig 5: p70 S6K is an essential target during JAGGED1-induced mineralization of HBO cells: HBO cells were treated with growth media alone, osteogenic media alone or with Fc-Dynabeads (5.7 μM), S6K-18 alone (a p70 S6K phosphorylation inhibitor) (50 μM), and JAG1-Dynabeads (5.7 μM) alone or in combination with S6K-18 (50 μM).The cells were half-fed every 5 days. On day 21 cells are fixed with 50% ethanol and thereafter, stained with 1% Alizarin Red S.(A) Representative image of HBO7. (B) Alizarin Red S dye was extracted from stained cells using a 1:10 dilution of acetic acid and water, and the absorbance was read at 420 nm. Data represent mean n = 3 ± standard deviation (SD) per cell line with p-values indicated.