Fig. 5

Pamidronate rescues the bilirubin-impaired dentinogenic capacity of SHED. a A schema of pamidronate treatment in bilirubin-impaired SHED under dentinogenic condition. SHED were cultured under dentinogenic condition stimulated with 0 μM bilirubin (B0), 50 μM bilirubin (B50), and 50 μM bilirubin plus 10 μM pamidronate (B50+PAM). b–d Calcium deposition assay after 4 weeks of the dentinogenic induction was performed by alizarin red (AR) staining. Representative images of AR staining were shown (b). AR-positive (AR⁺) area (c) and AR contents (d) in the cultures were measured. e–g Expression of odontoblast-specific genes and proteins after 2 weeks of the dentinogenic induction was analyzed. Relative expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), bone gamma-carboxyglutamic acid-containing protein (BGLAP), and dentin sialophosphoprotein (DSPP) genes to 18S rRNA (18S) was shown by real-time RT-PCR (e). Representative western blotting images of RUNX2, ALP, BGLAP, and DSPP were shown (f). Relative expression of RUNX2, ALP, BGLAP, and DSPP to beta-actin (ACTB) was analyzed (g). c–e, g n = 5 for all groups. Statistical analysis was performed as described in the “Methods” section. Graph bars showed the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.005. NS, no significance