Amelogenin differentially affects mineralization of cementoblasts and periodontal ligament cells

Objective:Enamel matrix derivative (EMD) is an acidic extract of extracellular enamel matrix, and suggested to exert various biological functions leading to periodontal tissue regeneration. Amelogenin is a major component of EMD, and exerts biological functions as signaling molecules through cell surface receptors. However, EMD also consists of heterogeneous mixture of mainly hydrophobic polypeptides, which may also have biological activity, suggesting that it is unclear which constituent of EMD induces regeneration and what molecular mechanism is involved. The purpose of this study was to examine differential effects of human full-length amelogenin on the mineralization activity of two-type periodontal tissue-derived cells. Methods:Recombinant human full-length amelogenin (rh174) was synthesized and purified. Cell lines of human cementoblasts (HCEM) and periodontal ligament fibroblasts (HPDL) established from periodontal tissue of a donor were used. Each cell line was cultured and treated with 0-1,000ng/mL rh174. The mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin (OCN) and bone sialoprotein (BSP) as mineralization markers were determined using quantitative real-time-PCR and Western blot analyses, respectively. ALP activity and calcium deposition were measured by ELISA. Mineralization was evaluated by Alizarin-Red Staining. Results:Gene expression levels of ALP, OCN, and BSP in HCEM were up-regulated by the treatment with rh174 in a dose-dependent manner. In addition, ALP activity and calcium concentration were enhanced significantly by the treatment with rh174, resulting in enhanced mineralization in HCEM. On the other hand, no significant effects of rh174 were shown on the mineralization in HPDL. Conclusion:It is shown that rh174 enhances mineralization accompanied by the up-regulation of mineralization markers in HCEM, but exerts no effects on the mineralization of HPDL, suggesting differential effects of amelogenin on the mineralization of periodontal ligament cells and cementoblasts. Such effects of amelogenin is suitable for the regeneration of periodontal tissues, which is equivalent to that achieved by mesenchymal stem cells.