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. 2025 Jul 23;11(30):eady2862. doi: 10.1126/sciadv.ady2862

Fig. 3. Supplementation of citrate, glutamine, and magnesium synergistically enhanced osteogenesis.

Fig. 3.

(A) ALP activity on day 7 of osteogenic differentiation with control, 200 μM citrate (CA), 200 μM citrate + 8 mM magnesium (CA + Mg), 200 μM citrate + 1.5 mM glutamine (CA + Gln), or 200 μM citrate + 1.5 mM glutamine + 8 mM magnesium (CA + Gln + Mg). Data are average ± SD. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 3. (B) mRNA expression of Runx2 on day 4 of hBM-MSCs osteogenic differentiation with CA or CA + Gln + Mg. Data are average ± SD. Two-tailed unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 3. (C) mRNA expression of Spp1, ALPL, and BMP2 on day 7 of hBM-MSCs osteogenic differentiation with CA or CA + Gln + Mg. Data are average ± SD. Two-tailed unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 3. (D and E) Representative images and quantification of calcium nodules deposition (red) with control, CA, CA + Mg, CA + Gln, or CA + Gln + Mg on day 14 of hBM-MSCs osteogenic differentiation. Scale bar, 500 μm. Data are average ± SD. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 3. (F and G) Representative images and quantification of ALP (green) immunofluorescence staining with DAPI nuclear counterstain (blue) on day 7 of hBM-MSCs osteogenic differentiation with control, CA or CA + Gln + Mg. Scale bar, 200 μm. (H and I) Representative images and quantification of OPN (red) immunofluorescence staining with DAPI nuclear counterstain (blue) on day 14 of hBM-MSCs osteogenic differentiation with control, CA, or CA + Gln + Mg. Scale bar, 200 μm. Data are average ± SD. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 3. OPN, osteopontin.