Prenylflavonoids isolated from Macaranga tanarius stimulate odontoblast differentiation of human dental pulp stem cells and tooth root formation via the mitogen‐activated protein kinase and protein kinase B pathways

Abstract

Aim To identify odontogenesis‐promoting compounds and examine the molecular mechanism underlying enhanced odontoblast differentiation and tooth formation. Methodology Five different nymphaeols, nymphaeol B (NB), isonymphaeol B (INB), nymphaeol A (NA), 3’‐geranyl‐naringenin (GN), and nymphaeol C (NC) were isolated from the fruit of Macaranga tanarius. The cytotoxic effect of nymphaeols on human DPSCs was observed using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The effect of nymphaeols on odontoblast differentiation was analyzed with Alizarin Red S staining and odontoblast marker expression was assessed using real‐time polymerase chain reaction and western blot analysis. The molecular mechanism was investigated with western blot analysis. In order to examine the effect of INB on dentine formation in the developing tooth germ, INB‐soaked beads were placed under the tooth bud explants in the collagen gel; thereafter, the tooth bud explant‐bead complexes were implanted into the sub‐renal capsules for 3 weeks. Tooth root formation was analyzed using micro‐computed tomography and histological analysis. Data are presented as mean ± standard error (SEM) values of three independent experiments, and results are compared using a two‐tailed Student's t‐test. The data were considered to have statistical significance when the p‐value was less than 0.05. Results Three of the compounds, NB, INB, and GN, did not exert a cytotoxic effect on human DPSCs. However, INB was most effective in promoting the deposition of calcium minerals in vitro (ppppin vivo (p<0.05). Conclusions Prenylflavonoids, including INB, exerted stimulatory effects on odontoblast differentiation and tooth root and dentine formation via the MAP kinase and AKT signaling pathways. These results suggest that nymphaeols could stimulate the repair processes for dentine defects or injuries.