Effects of Dentin Phosphophoryn-Derived RGD Peptides on the Differentiation and Mineralization of Human Dental Pulp Stem Cells In Vitro

Abstract

The purposes of this study were to investigate the in vitro effects of arginine-glycine-aspertic acid (RGD) peptides derived from human dentin phosphophoryn (DPP) on human dental pulp stem cell-proliferation, differentiation and mineralization, and to explore the mechanism of the peptides’ function. The 1 M concentration of soluble DPP-derived RGD peptides, RGD-1, RGD-2 and RGD-3 were coated onto non-tissue-culture polystyrene plates, and human dental pulp stem cells (hDPSCs) were cultured on them to examine the effects of the peptides on hDPSCs. In addition, 1 M arginine-alanine-aspertic acid (RAD) peptides were used as the control. Cell proliferation of hDPSCs was promoted by all three RGD peptides. All three RGD peptides had significantly higher alkaline phosphatase (ALP) activity compared to the control. RGD-3 induced the highest ALP activity compared to the control. RGD-3 also significantly promoted the mRNA expression of the following genes: 1.69-fold in dentine matrix protein-1 (DMP-1), 1.99-fold in dentine sialophosphoprotein (DSPP), 1.51-fold in ALP, and 2.31-fold in bone sialoprotein (BSP), as compared to the control group. Mineralization of hDPSCs was accelerated by all three RGD peptides, RGD-3 in particular. The MAPK p38 inhibitor SB202190 inhibited the effect of RGD-3 to a level comparable to the control, observed in both ALP activity assay and Arizarin red S (ARS) staining. It suggests that the p38 pathway may be responsible for eliciting the differentiation and mineralization effects of DPP-derived RGD peptides in the hDPSCs. The mRNA expression levels of the integrins ITGA1-5, ITGA7, ITGB1 and ITGB3 were significantly upregulated. Among them, expression of ITGA5 was promoted 1.9-fold, ITGA7 1.58-fold, ITGB1 1.75-fold and ITGB3 1.9-fold compared to the control. It suggests the possible involvement of these integrin channels in different subunit combinations facilitating signal transduction for differentiation of hDPSCs into odontoblasts. As conclusions, human DPP-derived RGD peptides RGD-1, RGD-2 and RGD-3 promoted the proliferation, differentiation and mineralization of hDPSCs in vitro. Among the three peptides, RGD-3 had the most significant effects. It is also suggested that RGD-3 binds to integrin receptors on the surface of hDPSCs and regulates the odontogenic gene expression and differentiation via activation of p38 of MAPK pathway. DPP-derived RGD-3 may be a promising choice in the formulation of a novel material for vital pulp therapy to induce dental pulp stem cells into odontoblasts and form reparative dentin on the exposed pulp tissue.

The odontoblastic differentiation of dental mesenchymal stem cells: molecular regulation mechanism and related genetic syndromes

“…In hDPCs, Estrogen 17β-estradiol (E2) increases the expression of odontoblastic differentiation markers, enhancing odontoblastic differentiation by activating c-Src and MAPKs ( Woo et al, 2015 ). The arginine-glycine-aspertic acid (RGD) peptide RGD-3 mediates odontogenic-related gene expression and enhances odontoblastic differentiation, during which the p38 MAPK pathway is activated in DPSCs ( Hassan et al, 2022 ). The insulin-like growth factor (IGF) family contributes to odontoblast differentiation via the MAPK pathway ( He et al, 2022 ).…”