Recombinant Amelogenin Regulates the Bioactivity of Mouse Cementoblasts in Vitro

Abstract

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL-1) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL-1 increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL-1) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL-1 remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL-1 rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue., A protein with its roots in dental development stimulates the proliferation and gene expression of cells linked to regeneration. Amelogenin is a mediator of enamel and tooth root formation, and the main component of a recently-developed medicine for periodontal regeneration. An international research group led by Sema Hakki, of Selcuk University, Turkey, has now elucidated the effects of amelogenin on cementoblasts, a type of cell responsible for producing the vital, mineralized layer on surface of the tooth root. Hakki’s team found that the bacteria-derived amelogenin increased the rate of mouse cementoblast proliferation and mineralization in vitro, and increased the expression of genes related to bone and tissue generation. The team also demonstrated the presence of a likely amelogenin receptor on the cells used in their study. These findings support further investigation into amelogenin’s therapeutic potential.