Effect of dentine phosphophoryn-derived RGD peptides on odontoblast-like cells.

Abstract

To investigate the effect of RGD peptides derived from dentine phosphophoryn (DPP) on odontoblast-like cell in terms of differentiation and mineralization. Mouse dental papilla cell line (MDPC-23), a rat odontoblast-like cell line, was used. Briefly, RGD peptides (RGD-1: SESDNNSSSRGDASYNSDES, RGD-2: ANSESDNNSSSRGDA, RGD-3: SRGDASYNSDESKD) were immobilized onto tissue culture polystyrene dishes (TCPS) assisted by carbodiimide chemistry. Surface characterization including carboxyl group quantification and amino acid analysis was carried out to ensure the existence of peptides on plates. Cells were inoculated to those peptides-modified and control dishes. Next, cell morphology was observed under phase contrast microscopy; cell numbers were counted manually using a hemocytometer. Furthermore, differentiation was examined by alkaline phosphatase (ALP) activity quantification, conventional and real-time RT-PCR. Finally, calcific deposition was observed by alizarin red staining and quantified using the cetylpyridinium chloride extraction method. Differences between the experimental groups and the control group were analysed statistically using one-way anova and Tukey's multiple comparison tests. Peptides were immobilized onto TCPS successfully as evidenced by carboxyl group density and amino acid analysis. Cell morphology remained unchanged between peptides-immobilized groups and control, but adhered cell numbers were higher on those peptides-immobilized dishes (significant differences existed between RGD-1-0.5 with control, RGD-2-0.1 with control, and RGD-3-0.5 with control, respectively). RGD-3-0.5 exhibited the highest ALP activity on day 7 (P<0.05) and promoted a twofold greater DMP-1 mRNA expression compared to the control on day 10 (P<0.05). RGD peptides grafted dishes accelerated the mineralization of cells, amongst the experimental groups tested, RGD-3 groups (comprising RGD-3-0.1 and RGD-3-0.5) had significantly higher amounts of calcific deposition as compared to the control (P<0.05). RGD peptides originated from DPP especially RGD-3 promoted MDPC-23 differentiation and mineralization.