Forskolin enhanced the osteogenic differentiation of human dental pulp stem cells in vitro and in vivo.

BACKGROUND/PURPOSE: Human dental pulp stem cells (hDPSCs) are multipotent adult stem cells that can differentiate into various lineages such as odontoblasts, osteoblasts, and chondrocytes. Regulation of hDPSCs differentiation with small-molecule compounds can be a useful tool for tissue engineering and regenerative therapy. Forskolin is an agonist of adenylate cyclase that promotes cyclic adenosine monophosphate production. However, the role of Forskolin in regulating the osteogenic differentiation of hDPSCs is still unknown.

MATERIALS AND METHODS

A cell counting kit-8 (CCK-8) assay was performed to screen out the safety concentrations of Forskolin. Following, quantitative polymerase chain reaction (qPCR) and alizarin red staining were performed to detect bone-related gene expression and mineralized deposit formation. Furthermore, we prepared cell sheets which were followed by a 3D culture for cell pellet formation. Finally, the hDPSC cell pellets were transplanted into immunodeficient mice.

RESULTS

CCK-8 assay showed 5 μM and 10 μM Forskolin had no significant inhibition on the proliferation of hDPSCs. The qPCR indicated Forskolin (5, 10 μM) enhanced osteogenic differentiation of hDPSCs by upregulating bone-related genes. Alizarin red staining and its quantification analysis demonstrated Forskolin in 5 μM and 10 μM similarly enhanced the mineralized deposit formation of hDPSCs in vitro. After six weeks of transplantation, immunohistochemical stains showed that osteopontin expression and bone formation were significantly boosted in the Forskolin-treated group than in the normal osteogenic inducing group.

CONCLUSION

Our results indicate Forskolin enhances osteogenic differentiation of hDPSCs in vitro and boosts bone formation in vivo.