Figure 5
Effects of DA on the odontoblastic differentiation of DPSCs-3U and on the reparative dentin formation. (A–C) DPSCs were cultured in DM containing with or without dopamine (0.1, 1, and 10 μM). (A) After 7 days of cultivation, calcium deposits were analyzed by Alizarin Red S staining and von Kossa staining. (B) Positive area of Alizarin Red S staining and von Kossa staining were quantified using a Keyence BZ-9000 microscope with BZ-H4M/BZ-H4C/BZ-H4CM software. (C) After 3 days of cultivation, the gene expression of DMP-1, DSPP, and NESTIN were investigated by quantitative RT-PCR. β-actin was used an internal control. Statistical analysis was performed using the Student’s unpaired or one-way ANOVA followed by Bonferroni’s test with Easy R software. t-test. n = 3, **P < 0.01, *P < 0.05. (D–U) The surface of exposed pulp was capped with nano β-TCP/collagen scaffold containing DA (D–F, K–U) or DW (G–I). (D–I) μCT images of the maxillary first molars at day 21 after direct pulp capping treatment with DA (D–F) or DW (G–I). (E, F) Magnified images of the red boxed areas in (D). (H, I) Magnified images of the red boxed areas in (G). (F, I) Newly reparative dentin was displayed in green. (J) The reparative dentin volume of these images was quantified using TRI/3D/VIE-FCS software. (K–M) HE staining of the maxillary first molars in DA treatment group. (K–U) Immunohistochemical staining of the maxillary first molars in DA treatment group with anti-Nestin antibody (N–P) and anti- Th antibody (Q–S). (L, O, R) Magnified images of the black boxed area in (K, N, Q). (M, P, S) Magnified images of the dotted black boxed area in (L, O, R). (T, U) Rabbit control IgG was used as a negative control. (U) Magnified images of the black area in (T). Nuclei were stained with hematoxylin. Bars = 100 µm. Arrowheads; dentinal tubules. Statistical analysis was performed using the Student’s unpaired test with Easy R software. t-test. n = 6, **P < 0.01.
