Synthetic Ameloblastin Peptide Stimulates Differentiation of Human Periodontal Ligament Cells

Objective: This study investigates the effect of the N-terminal region of a synthetic porcine ameloblastin peptide on the proliferation and differentiation of human periodontal ligament cells (PDLC). Methods: An ameloblastin peptide, based on the 16 amino acid sequence of the N-terminal porcine ameloblastin (VPAFPRQPGTPGVASL), was synthesized. We used a cell counter to assess the effect of ameloblastin peptides on the proliferation of PDLC. To investigate the effect of ameloblastin peptides on the differentiation of PDLC, quantitative analysis of alkaline phosphatase (ALP) activity by the Bessey-Lowry enzymological method, mineral nodule formation by Dahl's method, and expression of mineralization-related genes by RT-PCR were examined. We used an anti-ameloblastin antibody to determine whether stimulation of ALP activity was caused by the peptide specifically. Results: At all concentrations examined, the effect of the ameloblastin peptide on cell proliferation was not significantly different compared with the control. However, the peptide significantly stimulated ALP activity in a dose-dependent manner. ALP activity was significantly inhibited by the anti-ameloblastin antibody, which caused ALP levels to revert to the approximate levels in the untreated condition. At concentrations greater than 1 ng/ml, the peptide promoted mineralized nodule formation of PDLC. At 10 and 100 ng/ml, the expression of bone sialoprotein (BSP) was higher than that of control cells. Conclusion: Our results show that the ameloblastin peptide upregulates ALP and BSP levels and can enhance calcification of PDLC. Thus, we suggest that the N-terminal synthetic ameloblastin peptide may be a useful therapeutic agent for regenerating periodontal tissue.