Fig. 2.

The osteopromotive effect of solute citrate. (A) Gene expression of Runx2, COL1a1, and SPP1 in differentiating hMSCs with/without citrate supplementation at 200 µM in established OG medium for 7 and 14 d, as determined by real-time PCR. Data are shown as the expression of target genes after normalization relative to corresponding control groups without citrate supplementation at day 7 (value set to 1.0). (B) Expression of Runx2 transcription factor in hMSCs with/without citrate supplementation in OG medium as determined by ELISA. In A and B, n ≥ 4 biological replicates per group. (C) Schematic illustration of the experimental design studying differentiation-stage dependence by restricting citrate supplementation (200 µM) only at the proliferation stage (group I; days 0–4), the matrix maturation stage (group II; days 4–14), the mineralization stage (group III; days 14–21), or after 4 d in GM before osteoinduction (group IV; day −4 to day 0). (D) ALP expression at day 14 and calcium content in hMSCs at day 21 in OG medium supplemented with citrate only at specific differentiation stages (groups I, II, and III). (E) ALP production and calcium content in hMSCs with/without 4 d of citrate pretreatment in GM before being subjected to osteogenic differentiation in OG medium without citrate addition (group IV). In D and E, n = 3–5 biological replicates per group. All data are presented as mean ± SD; *P < 0.05.