Amphiregulin regulates odontogenic differentiation of dental pulp stem cells by activation of mitogen-activated protein kinase and the phosphatidylinositol 3-kinase signaling pathways.

BACKGROUND: Human dental pulp stem cells (hDPSCs) have received widespread attention in the fields of tissue engineering and regenerative medicine. Although amphiregulin (AREG) has been shown to play a vital function in the biological processes of various cell types, its effects on DPSCs remain largely unknown. The aim of this study was to explore the specific role of AREG as a biologically active factor in the regeneration of dental pulp tissue. METHODS: The growth of hDPSCs, together with their proliferation and apoptosis, in response to AREG was examined by CCK-8 assay and flow cytometry. We explored the effects of AREG on osteo/odontogenic differentiation in vitro and investigated the regeneration and mineralization of hDPSCs in response to AREG in vivo. The effects of AREG gain- and loss-of-function on DPSC differentiation were investigated following transfection using overexpression plasmids and shRNA, respectively. The involvement of the mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)/Akt pathways in the mineralization process and the expression of odontoblastic marker proteins after AREG induction were investigated by using Alizarin Red S staining and Western blotting, respectively. RESULTS: AREG (0.01-0.1 µg/mL) treatment of hDPSCs from 1 to 7 days increased hDPSCs growth and affected apoptosis minimally compared with negative controls. AREG exposure significantly promoted hDPSC differentiation, shown by increased mineralized nodule formation and the expression of odontoblastic marker protein expression. In vivo micro-CT imaging and quantitative analysis showed significantly greater formation of highly mineralized tissue in the 0.1 µg/mL AREG exposure group in DPSC/NF-gelatin-scaffold composites. AREG also promoted extracellular matrix production, with collagen fiber, mineralized matrix, and calcium salt deposition on the composites, as shown by H&E, Masson, and Von Kossa staining. Furthermore, AREG overexpression boosted hDPSC differentiation while AREG silencing inhibited it. During the differentiation of hDPSCs, AREG treatment led to phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and PI3K/Akt. Notably, a specific inhibitor of ERK, JNK, and PI3K/Akt signaling markedly reduced AREG-induced differentiation, as well as levels of phosphorylated ERK and JNK in hDPSCs. CONCLUSIONS: The data indicated that AREG promoted odontoblastic differentiation and facilitated regeneration and mineralization processes in hDPSCs.